Received 16 December 2005/Returned for modification 6 February 2006/Accepted 6 May 2006

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 2006, p Vol. 44, No /06/$ doi: /jcm Copyright 2006, American Society for Microbiology. All Rights Reserved. Detection of Chlamydia trachomatis by Nucleic Acid Amplification Testing: Our Evaluation Suggests that CDC-Recommended Approaches for Confirmatory Testing Are Ill-Advised Julius Schachter, 1 * Joan M. Chow, 2 Holly Howard, 2 Gail Bolan, 2 and Jeanne Moncada 1 Chlamydia Research Laboratory, Department of Laboratory Medicine, University of California, San Francisco, California, 1 and Sexually Transmitted Disease Control Branch, California Department of Health Services, Richmond, California 2 Received 16 December 2005/Returned for modification 6 February 2006/Accepted 6 May 2006 We evaluated three CDC-suggested approaches for confirming positive nucleic acid amplification tests (NAATs) for Chlamydia trachomatis: (i) repeat the original test on the original specimen, (ii) retest the original specimen with a different test, and (iii) perform a different test on a duplicate specimen. For approach 1, specimens (genital swabs or first-catch urine [FCU]) initially positive by the Abbott LCx Probe System Chlamydia trachomatis Assay (LCx; Abbott Laboratories), the APTIMA Combo 2 Assay (AC2; Gen-Probe Inc.), the Amplicor CT/NG Assay (PCR; Roche Diagnostics Corp.), or the BD ProbeTec ET System C. trachomatis amplified-dna assay (SDA; Becton Dickinson Diagnostic Systems) were retested by the same NAAT. In several evaluations, multiple efforts were made to confirm the original positive result. For approach 2, specimens initially positive by SDA and the Hybrid Capture 2 CT-ID DNA Test (HC2; Digene Corp.) were retested by different NAATs (SDA, PCR, AC2, and the APTIMA assay for C. trachomatis [ACT]). For approach 3, duplicate male urethral or cervical swabs were tested by SDA or by both AC2 and ACT. FCU specimens were tested by all three tests. We found that 84 to 98% of SDA, LCx, PCR, and AC2 positive results were confirmed by a repeat test and that 89 to 99% of SDA and AC2 and 93% of HC2 positive results were confirmed by different NAATs, but that some NAATs cannot be used to confirm other NAATs. The use of repeat testing did not confirm 11% of C. trachomatis SDA positive results that could be confirmed by more extensive testing. Doing more testing confirms more positive results; >90% of all positive NAATs could be confirmed. Nucleic acid amplification tests (NAATs) are the most sensitive tests for screening and diagnosis of chlamydial and gonococcal infections of the genital tract (1, 4, 10, 11, 15, 20 22). However, questions have been raised about their specificity, particularly when screening low-prevalence populations (3, 5, 12). In 2002, these concerns led the Centers for Disease Control and Prevention (CDC) to recommend confirmatory testing of positive NAATs for Chlamydia trachomatis when the positive predictive value (PPV) of a test is 90% (9). The assumptions are that failure to confirm a positive result means the initial positive result was likely a false positive and that confirming the initial positive result increases confidence that it was a correct result. However, the premise that failure to repeat the initial positive result means that it was a false positive may not be true. Package insert specificities range from 97.6 to 98.9% for these tests. However, these specificities are not based on discrepant analysis, because statisticians have criticized that method (6, 16, 19). The true specificities of NAAT methods are in fact much higher, and several studies have found specificities to be 99% (1, 4, 8, 22). With a wide range of reported specificities and no guidance from the CDC, laboratories must decide which specificities are valid. If product insert specificities are accepted, then by CDC recommendations, all positive * Corresponding author. Mailing address: Chlamydia Research Laboratory, Department of Laboratory Medicine, University of California, San Francisco, 1001 Potrero Avenue, Bldg. 30 Room 416, San Francisco, CA Phone: (415) Fax: (415) Julius.Schachter@ucsf.edu. specimens in the low- to moderate-prevalence range (1 to 7%) would need confirmatory testing because PPVs are 90%. The CDC has suggested several possible strategies for confirmation, but it is not clear which approach is best. The proposed strategies include (i) testing a second specimen with a different test or target, (ii) performing a second NAAT targeting a different nucleic acid sequence on the original specimen, (iii) repeating the original test on the original specimen, and (iv) bringing the patient back for a retest. These approaches have limitations: (i) most clinicians will not routinely collect two specimens for the same evaluation, nor is it feasible to bring the patient back to collect another sample, and (ii) most laboratories do not have the capability to perform two different NAAT methodologies. Currently, the approach of repeat testing is the only feasible confirmatory test available to most laboratories. The concept of confirmatory testing is not new (2, 5, 12). In February 2001, following reports of low test performance and quality control issues with their test (5), Abbott Laboratories suggested that all positive patient samples in their LCx Probe System Chlamydia trachomatis Assay should be confirmed by a repeat test. Eventually, the product was discontinued, but many laboratories switched to other NAATs and continued to retest positive results as part of their standard operating procedure. From 2001 to 2004, we performed confirmatory nucleic acid amplification testing, using different strategies. Here, we report our experiences when confirming positive results for C. trachomatis by repeating the original test on the original specimen, by testing the original specimen with a different NAAT, and by testing duplicate specimens. 2512

2 VOL. 44, 2006 POSITIVE C. TRACHOMATIS NAAT CONFIRMATION IS ILL-ADVISED 2513 MATERIALS AND METHODS NAATs evaluated. We evaluated the following NAATs: the LCx Probe System Chlamydia trachomatis Assay (LCx; Abbott Laboratories, Abbott Park, IL), the APTIMA Combo 2 Assay and APTIMA assay for C. trachnomatis (AC2 and ACT; Gen-Probe Inc., San Diego, CA), the Amplicor CT/NG Assay (PCR; Roche Diagnostics Corp., Indianapolis, IN), and the BD ProbeTec ET System C. trachomatis amplified-dna assay (SDA; Becton Dickinson Diagnostic Systems, Sparks, MD). The two APTIMA format tests target different rrna sequences. All of the tests except LCx are commercially available. In addition, a DNA probe test with signal amplification (18), the Hybrid Capture II CT-ID DNA Test (HC2; Digene Corp., Gaithersburg, MD) was evaluated. We followed package insert instructions for the initial testing of clinical specimens. Specimens were collected in the NAAT s recommended transport medium unless otherwise indicated. For confirmatory testing, we used the manufacturer s instructions for testing, but because each manufacturer supplies a different transport medium, we had to vary the processing of the specimens as discussed below. Specimen types. For women, we tested first-catch urine (FCU) and endocervical swabs. For men, we tested FCU and urethral swabs. For men who had sex with men, we also tested rectal and pharyngeal swabs, specimens not FDA cleared for NAATs. All specimens were initially tested by a NAAT for C. trachomatis, and positive specimens were saved for confirmatory testing. If retested within 48 h, specimens were stored at 4 C. If not, then the specimens were stored at 70 C. Approach 1: repeat testing for confirmation of positive results. Here and elsewhere, where a subset analysis is mentioned, there was neither randomization nor specific selection criteria (see below for reasons) for specimens for a retest. (i) LCx. From February 2001 to November 2002, routine clinical specimens were initially tested by LCx. All positive specimens were repeat tested by LCx. On a subset of these positive results, the original LCx samples were confirmed by PCR (250 l of the LCx sample was spun at 12,000 g for 15 min, the supernatant was discarded, and the protocol for PCR swab processing and testing was followed). In addition, LCx positive specimens with initial low-level ( 1.0 but 1.9) sample rate/cutoff ratios were retested repeatedly (eight times) in different LCx assay runs. AC2. From June 2003 to July 2004, clinical specimens were initially tested by the AC2 test. All positive specimens were repeat tested by AC2. The original positive specimens were then confirmed by the ACT assay. In addition, AC2 positive specimens with initial low-level readings were retested on six separate assay runs. (ii) PCR. From June 2003 to March 2004, some clinical specimens were initially tested by PCR, and positive specimens were repeat tested by PCR. (iii) SDA. From June 2003 to August 2004, some clinical specimens were initially tested by SDA. Positive specimens were repeat tested by SDA twice. In addition, positive specimens with initial low-level method other than acceleration (mota) readings were retested on four separate assay runs. (iv) Repeat testing for specificity. With each assay (LCx, SDA, and AC2), a subset of specimens with an initial negative C. trachomatis result was retested repeatedly by the same assay in different assay runs. Approach 2: use of a different NAAT for confirmation. (i) Study 1. From January 2003 to September 2004, a moderate-prevalence population was tested for C. trachomatis. Swabs and FCU were collected from men and women seen in obstetrics/gynecology (OB/GYN) clinics, family planning clinics, or a sexually transmitted disease (STD) clinic in the San Francisco Bay area. Specimens were collected in BD s ProbeTec transport tubes and originally tested by SDA. C. trachomatis-positive specimens were repeat tested by SDA, and then aliquots of the original specimens were tested by PCR and AC2. For PCR confirmation, 500 l of the SDA sample was spun at 12,000 g for 15 min, the supernatant was discarded, and the protocol for PCR swab processing and testing was followed. For AC2 confirmation, 100 l of the SDA sample was inoculated into an AC2 swab transport tube and tested according to the package insert instructions. (ii) Study 2. From May 2003 to June 2004, a low-prevalence population was screened for C. trachomatis. Swabs and FCU were collected from women seen in OB/GYN clinics, family planning clinics, and an HMO Kaiser Permanente Group in Southern California. The great majority of the patients were asymptomatic and 21 years old. Specimens were originally tested by SDA. All positive specimens were held at 70 C until they were shipped to the University of California, San Francisco, Chlamydia Research Laboratory. Upon receipt, the samples were thawed and retested by SDA, as well as PCR, AC2, and ACT. For PCR, AC2, and ACT confirmation, the SDA sample was treated as described above. A separate 400- l aliquot was tested with the AC2 and ACT assays. TABLE 1. Approach 1: confirmation of positive NAAT results for C. trachomatis Initial test No. positive Confirmatory test % Confirmed LCx 251 LCx PCR 93.3 AC2 217 AC ACT ACT AC PCR 30 PCR 96.7 SDA 80 SDA 83.8 SDA twice 92.5 (iii) Study 3. From October 2003 to June 2004, a low-prevalence population for C. trachomatis was screened in OB/GYN clinics in southern California. The majority of patients were asymptomatic. Cervical brushes were collected in PreservCyt (ThinPrep Pap Test; Cytyc Corp., Boxborough, MA) and tested by HC2 for C. trachomatis. All positive specimens were held at 70 C until they were shipped to the University of California, San Francisco, Chlamydia Research Laboratory. Upon receipt, the samples were thawed and tested by SDA, PCR, AC2, and ACT. For SDA confirmation, 4.0 ml of the positive PreservCyt was spun at 2,000 g for 30 min, the supernatant was discarded, and the protocol for swab processing and testing was followed. For PCR confirmation, 500 l of the positive PreservCyt was spun at 12,000 g for 15 min, the supernatant was discarded, and the protocol for swab processing and testing was followed. For AC2 and ACT confirmation, 100 l of the positive PreservCyt was inoculated into an AC2 swab transport tube. A separate 400- l aliquot was tested with the AC2 and ACT assays. Approach 3: testing a second specimen with a different test or target for confirmation. The study using approach 3 was recently published (17). Briefly, from January 2003 to May 2003, a multicenter clinical evaluation of the AC2 and ACT assays was conducted in North America. Subjects attended STD, OB/GYN, teen, or family planning clinics in seven geographically diverse centers. There were 1,465 females and 1,322 males enrolled. FCU and two randomized endocervical swabs were collected from females. FCU and two randomized urethral swabs were collected from males. Each specimen type was tested by ACT, AC2, and SDA. Thus, three NAATs, each having a different target, were used to test specimens for each subject. The SDA, ACT, and AC2 assays were each considered the primary screening tests, and all positive results for each test were then confirmed by both of the other tests. RESULTS Table 1 shows results for C. trachomatis confirmation by repeating the original test on the original specimen. The overall chlamydia prevalence was 5% for the reproducibility studies. AC2 and PCR repeated 97.7% and 96.7% of positive results, respectively. SDA confirmed only 83.8% by repeat testing. However, by testing SDA positive specimens a third time, an additional 9% were confirmed. LCx high-level positive results seldom failed to repeat; failure to retest positive was associated with specimens having low (1.0 to 1.9) sample rate/ cutoff ratios, where 53% of the positive results did not repeat (Fig. 1). Similarly, 58% of low-level SDA positive (2,000 to 10,000) mota readings did not repeat, but failures were seen in all ranges, even at the high end of 40,000 mota (Fig. 2). When low-level LCx, SDA, and AC2 positive specimens were retested several times, variable results were observed (Table 2). Each positive result was ultimately confirmed, but an apparently random sequence of positive and negative results was seen with the retests. In two of these repeat studies (LCx and AC2), we were also able to use another NAAT for confirmation. Using PCR on LCx positive specimens failed to confirm

3 2514 SCHACHTER ET AL. J. CLIN. MICROBIOL. FIG. 1. Distribution of LCx positive results that failed to repeat (n 24). S/C, sample rate/cutoff. some specimens that repeat testing confirmed (Table 1). By using ACT to confirm AC2 (Table 1), confirmation was slightly better than by repeat testing (99.1% versus 97.7%). Of the AC2 repeat failures, four of five samples had low relative light unit readings ( 500,000). Three of the NAATs were evaluated for reproducibility of negative results. Table 3 shows that LCx, SDA, and AC2 had 99.5% specificities, assuming the original negative result was correct. Of the initially negative AC2 specimens, two showed multiple positive results in the five repeat tests (Table 2). For study 1, approximately 1,932 patients were screened. The majority were female; 21% were male. The overall prevalence of C. trachomatis was 6.7%. There were a total of 130 SDA C. trachomatis original positive results (Table 4), of which 87.7% repeat tested as positive. When different NAATs were used for confirmation, PCR confirmed 83.8% while AC2 confirmed 86.9%. However, by using a combination of repeat SDA, PCR, or AC2, 94.6% of the original positive results were confirmed. For study 2, we screened 4,961 patients with an overall C. trachomatis prevalence of 3.5%. There were a total of 111 SDA C. trachomatis original positive results (Table 4). We found that only 79.3% repeat tested positive (P 0.077) compared to study 1. When different NAATs were used for confirmation, PCR confirmed 73.9%, while AC2 and ACT both confirmed 81.1%. However, by using different combinations of confirmatory tests, more positive results (90.1%) were confirmed. In study 3, the HC2 assay identified a C. trachomatis prevalence of 3.4% in the 2,673 patients screened. Of the 90 HC2 positive specimens, 72 were evaluated. Using different NAATs for confirmation, SDA, PCR, and ACT confirmed 90.3%, while AC2 confirmed 88.9%. However, by using a combination of three NAAT tests, 93.1% of the original HC2 positive results were confirmed. Table 5 shows our results using either a second specimen with a different NAAT for confirmation or a second NAAT on the same specimen (17). For all specimen types, AC2 had 850 positive results, the ACT assay had 927 positive results, and the SDA had 720 positive results. Both the ACT and AC2 confirmed 96.9% of the SDA positive results. Of the AC2 positive specimens, 98.1% were positive by ACT, but only 82% were positive by SDA. Of the ACT positive results, AC2 confirmed 89.8%, but the SDA test confirmed only 75.1%. There were no major differences in the observed patterns by sex or specimen type. SDA consistently confirmed fewer of the initially positive FIG. 2. Distribution of SDA positive results that failed to repeat (n 39). AC2 and ACT results in a second specimen or in the same FCU specimen. DISCUSSION We have found that the approaches for repeat testing for C. trachomatis confirmation vary in performance by the population tested and the number of retests done. In a C. trachomatis moderate-prevalence population, 90.4 to 97.7% of LCx, PCR, and AC2 positive results were confirmed by repeat testing using the same test (Table 1). Recently, a study reported that 93.7% of initial positive SDA results were confirmed with retesting under 8.1% C. trachomatis prevalence (22). We found that only 79.3% and 87.7% of initial SDA positive results were confirmed by retesting under low (3.5%) and moderate (6.7%) C. trachomatis prevalences (Table 4). Retesting low-level positive results may yield fluctuating positive and negative results (Table 2). It is not surprising that 50% of low-level LCx and SDA positive results were repeat failures. This is likely due to the small amount of target present and sampling variability consistent with Poisson s distribution. Failure to repeat a positive test does not mean the initial result was a false positive. A single repeat test would not confirm 6.9 to 10.8% of the total number of C. trachomatis SDA positive results that are confirmed by more extensive testing (Table 4) and 1.8% of C. trachomatis AC2 positive results that are confirmed by ACT testing (Table 1). It would seem, then, that much of the problem with reproducibility of positive NAAT results actually reflects the exquisite analytic sensitivity of these assays. It would be useful if there was an accepted international Chlamydia standard that laboratories could use to perform interassay evaluations and, through dilution studies, better understand the vagaries of testing near the assay detection limits. The enhanced analytic sensitivity of the NAATs also means that minor variations in processing could impact results. More emphasis should be placed on quality control and proficiency testing. Specimen adequacy could be an issue, and we might benefit from research on the possible benefits of including assays for human housekeeping genes in these diagnostic tests. The reproducibility of C. trachomatis-negative NAAT specimens was evaluated. Table 3 shows high reproducibility of

4 VOL. 44, 2006 POSITIVE C. TRACHOMATIS NAAT CONFIRMATION IS ILL-ADVISED 2515 TABLE 2. Reproducibility of results for Chlamydia-positive specimens a Run LCx result (S/C b ) for specimen: SDA result (mota value) for specimen: AC2 (RLU reading) for specimen: FPC8001 D0649 NGHC02 WK0078 X8 X31 X61 X b b Seeded c ,886 2,923 10,246 11, , , ,205 2, , a Values in boldface are for specimens that tested negative in the run. A positive LCx result was defined as a sample/cutoff ratio (S/C) of 1.0. A positive SDA result was defined as a mota value of 2,000. A positive AC2 result was defined as a relative light unit (RLU) reading of 100. b The original result was negative. c This specimen was seeded with a dilution of C. trachomatis and was expected to yield a low-level-positive result. negative results for LCx, SDA, and AC2, all 99.5%. These results are similar to C. trachomatis NAAT specificities found in many clinical evaluations using discrepant analysis. They are higher than the specificities listed in the package insert. The CDC-suggested approach of performing a second NAAT targeting a different nucleic acid sequence on the original specimen for C. trachomatis confirmation also varies in performance. Although NAATs are highly sensitive, there are differences among them. Some are more sensitive than others. For both SDA confirmation studies using a different test, PCR confirmed fewer positives than repeat testing, whereas AC2 confirmation was similar to that of repeat testing. With a moderate C. trachomatis prevalence, AC2 confirmed 86.9% of SDA positive results (Table 4). However, in evaluation of a different NAAT with a second specimen, we found that AC2 confirmed 96.9% of the SDA positive results (Table 5). Here, the higher number of positives confirmed was probably due to the methodology, as the collection of a second specimen in the recommended NAAT transport medium reflects more optimal testing conditions. Although our procedure using the original SDA transport medium was validated with AC2, ACT, and PCR, it may not be the most sensitive. PreservCyt transport medium was used for HC2 specimens, and all the NAATs had similar confirmation rates of 90%. By using a combination of three NAAT results, 93.1% of HC2 positive results were confirmed. In the evaluation of the third approach, it was clear that some NAATs cannot be used to confirm positive results from other NAATs because of differences in sensitivity (17). The use of the less sensitive SDA to confirm AC2 or ACT positive results would have resulted in wrongly reporting 15% of the confirmable ACT results as negative (i.e., not confirmed). When the SDA assay was used for the primary screening, both the AC2 and ACT assays confirmed 96.9% of the results. Thus, TABLE 3. Specificities of Chlamydia LCx, SDA, and AC2 assays inferred by repeat testing of initially negative specimens NAAT No. of specimens No. of repeats Specificity (%) LCx /512 (99.6) SDA /531 (99.6) AC /552 (99.5) when attempting to confirm positive NAAT results, the assays employed should have equivalent sensitivities. We have shown that all three of the CDC-suggested approaches for confirmation vary in performance. The use of just one approach would fail to confirm many positives. By using two of the approaches (repeat testing with the same test and with a different NAAT), we found 90.1 to 94.6% of SDA positive results were confirmed and 99.5% of AC2 positive results were confirmed. With 90% of our original C. trachomatis-positive results being confirmed in all the assays, we found that confirmatory testing of positive NAAT specimens is not necessary. This does not imply that false-positive results do not occur. They do, but at a very low rate, and confirmatory testing simply is not the way to identify them. The logic behind confirmatory testing is based upon two assumptions. The first is that if a positive result is repeated, it is likely to be a true positive. The underlying assumption here is that false-positive results will not be confirmed. It is unclear why this should be true, particularly when the same test or target is used. We do not have sufficient experience with proven false-positive specimens to evaluate this assumption, but logic says that it need not be true; if there is a compound capable of causing a false-positive result in the specimen, why should it not be there for the retest? The second assumption is that failure to repeat an initial positive result means the initial result was incorrect (i.e., a false positive). That assumption is demonstrably false. We found TABLE 4. Confirmation of positive SDA tests for C. trachomatis Confirmatory test Total no. (%) confirmed Study 1 a Study 2 b Initial test positive Repeat SDA (twice) 114 (87.7) 88 (79.3) PCR 109 (83.8) 82 (73.9) AC2 113 (86.9) 90 (81.1) ACT ND c 90 (81.1) Combined PCR/AC2 121 (93.1) 94 (84.7) Combined SDA/PCR/AC2 (twice) 123 (94.6) 99 (89.2) Combined SDA/PCR/AC2/ACT (twice) ND 100 (90.1) a Moderate prevalence. b Low prevalence. c ND, not done.

5 2516 SCHACHTER ET AL. J. CLIN. MICROBIOL. TABLE 5. Chlamydia-positive results obtained by NAAT confirmed by testing of a second specimen by other NAATs a Specimen type and initial test Gender No. of samples % Positive by confirmatory test AC2 ACT SDA First-catch urine AC2 Male Female Total b 85.3 ACT Male Female Total c 79.0 SDA Male Female Total Swab AC2 Male Female Total d 78.9 ACT Male Female Total e 71.5 SDA Male Female Total All AC2 Male Female Total b 82.0 ACT Male Female Total b 75.1 SDA Male Female Total a Adapted from reference 17. b The P value was for the difference between percentages c The P value was for the difference between percentages d The P value was for the difference between percentages e The P value was for the difference between percentages that the set of specimens in which an initial result was positive and a second test was negative had a higher prevalence of positive results on further testing than any incoming set of specimens (i.e., approximately 65% of these specimens could be shown to be positive in the third or subsequent tests). It is clear that some of these results reflect heterogeneity of these specimens (i.e., consistent with a Poisson distribution, where a single target is the threshold level of analytic sensitivity for the specific test). If on average the amount of target present in that specimen is at that level, then a large fraction (as high as 35%) of aliquots from the specimen would test negative. Certainly, we saw something like that happening with both clinical specimens and seeded specimens (Table 2). Thus, the initial positive result most likely reflects truth (i.e., the target was present in the original specimen, and the failure to verify was because there was too little target homogeneously distributed in the specimen to yield positive results in every subsequent test). These results have implications beyond testing for Chlamydia trachomatis, or for that matter, Neisseria gonorrhoeae (for which we had a similar experience with confirmatory testing with AC2 and SDA, but not with PCR). There will probably be similar results with efforts to confirm positive results in NAATs for other organisms. There are other implications. The variations we saw in test results are probably reproduced when multiple specimens are collected at different times from individuals with low-level infections. In other words, sampling over different time periods could result in intermittent negatives, and that may be misinterpreted as reflecting spontaneous clearance of infection. Rather than clearing of the infection, the negative result may reflect sampling variability or simply low target levels (in the NAAT sense). Years ago, when we evaluated the sensitivity of culture and did not have a more sensitive assay available, we estimated that the sensitivity of culture was on the order of 80% based on repeat testing of individuals prior to their being treated (14). Individuals who initially tested positive would test negative at the second visit and then positive at a third visit. Most of these data were developed during prospective studies of the risk of infection to newborns passing through an infected birth canal. The pregnant women were being seen on a regular basis, and those who were infected were often not found to be positive in isolation attempts during each visit. We assumed that with the use of the more sensitive NAATs, this sporadic positivity seen with culture would not be seen in these more sensitive assays. Our results suggest that is not likely to be the case. Thus, the failure to repeat positive results when rescreening individuals should not easily be accepted as spontaneous clearance; it could reflect the variability inherent with low copy numbers of targets. There is another implication of our results. If low-level positive results give variable results on repeat testing, it implies that some positive specimens will give negative results on the first test. Thus, if we retest initially negative specimens, we should get some that yield positive results on the repeat test. We did see some such results, but the sporadic nature of the positive results was impossible to interpret as anything but an occasional false-positive when we repeated the LCx and SDA assays. However, when we repeat tested initially negative AC2 specimens, a few (2/92) yielded the on/off pattern that we also observed with seeded specimens and with some initially positive specimens (Table 2). This suggests that there is still room for improving the sensitivity of NAATs. The variability could be due to minor technical variations due to the manual processing of specimens, irregular and less than optimal extraction of nucleic acid from the original specimen, or variability in the amplification (7, 13). Our results lead to several conclusions. (i) Confirmatory testing is unnecessary; it complicates the handling of a positive NAAT result, as the initial failure to confirm a positive result is wrong more often than it is right, and adds cost to an already expensive screening test. (ii) Given that we can confirm 90% of positive NAAT results when calculated PPVs are 90% using specificity figures from the package inserts, the real NAAT specificity must be better than the levels given in the package inserts. (iii) There is still room for improving the sensitivity of NAATs, perhaps by better specimen preparation, automation, or target concentration.

6 VOL. 44, 2006 POSITIVE C. TRACHOMATIS NAAT CONFIRMATION IS ILL-ADVISED 2517 ACKNOWLEDGMENTS This work was supported by the donation of test kits used for the confirmation of positive specimens by the following manufacturers: Abbott Laboratories, Becton Dickinson Diagnostic Systems, Gen- Probe Inc., and Roche Diagnostics Corp. Some of the data collection and specimen processing was done by Planned Parenthood Santa Barbara, Ventura, and San Luis Obispo Agency; Planned Parenthood Mar Monte Agency; Quest Diagnostics/Unilab; Medical Group Pathology Laboratory of Santa Barbara; and California Family Health Council. This study was partly supported by funds from the CDC, Atlanta, Ga. (STD Program Grant H25/CCH ). Human subject approval for study 1 was by the University of California, San Francisco, Committee on Human Research. Human subject approval for study 2 was by the University of California, Berkeley, Committee on Human Research. REFERENCES 1. Black, C. M., J. Marrazzo, R. E. 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