Impact of Patient Characteristics on Performance o
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微生物临床杂志 2005年第2期
Department of Medicine, University of Washington, Seattle, Washington
Division of STD Prevention, National Center for HIV, STD, and TB Prevention
National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Department of Laboratory Medicine, University of California
San Francisco Department of Public Health, San Francisco, California
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
School of Medicine, Indiana University, Indianapolis, Indiana
School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana
ABSTRACT
The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women 20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women.
INTRODUCTION
Genital infection with Chlamydia trachomatis is the most commonly reported bacterial disease in the United States and has well-established adverse effects on the reproductive health of women (4, 23). Nucleic acid amplification tests (NAAT), including PCR, ligase chain reaction (LCR), strand displacement assay, and transcription-mediated amplification, are more sensitive for the diagnosis of C. trachomatis than culture, nonamplified DNA probe, and antigen detection tests and maintain relatively high specificity (1, 2). Equally important, the ability to perform NAAT on urine and self-collected vaginal swabs has expanded opportunities for screening men and women (7, 10, 11, 14, 21, 26, 28, 29).
Our understanding of the clinical manifestations of chlamydial infections in women, including endocervical and urethral inflammation, has been based largely on studies using non-NAAT for detection of infection. Similarly, selective screening criteria for identifying cervical chlamydial infection have been based on risk factors as defined by various non-NAAT. NAAT, however, can detect the presence of fewer chlamydial elementary bodies than non-NAAT (1). Accordingly, among men with urethral chlamydial infection, NAAT demonstrate the greatest increase in sensitivity over non-NAAT in asymptomatic men without urethral inflammation (22). One possible explanation for this is that the quantity of elementary bodies appears to correlate directly with signs of inflammation in men and women and with the presence of symptoms in men (12, 22). Whether the increase in sensitivity of NAAT over non-NAAT is greatest among women with no signs of endocervical or urethral inflammation is not known.
Some data also suggest that NAAT may be inhibited by substances present in clinical specimens, including blood, beta human chorionic gonadotrophin, and products of inflammation (6, 15, 19, 27). However, few studies have directly evaluated whether the performance of NAAT in clinical settings is affected by menstrual blood or by endocervical or urethral inflammation. We recently published an analysis of the performance of NAAT versus an unamplified DNA probe using an expanded and independent diagnostic reference standard in a large, diverse population of women seeking care for family planning or sexually transmitted disease (STD) concerns at five centers across the United States (2). The present study expands these analyses to evaluate the impacts of other factors, including age, endocervical and urethral inflammation, and menstrual blood, on the performance of these tests.
MATERIALS AND METHODS
Women who presented to an STD clinic in each of five participating centers and one family planning clinic who were 16 years old and not pregnant and had not taken antibiotics in the previous 30 days were eligible for enrollment, regardless of genitourinary symptoms. Study clinicians informed eligible patients about the study and enrolled those who gave informed consent. The study was approved by the institutional review board at each center and at the Centers for Disease Control and Prevention. Enrollment began in October 1995 and ended in August 1997, when the target sample of 407 women with urethral or endocervical cultures positive for C. trachomatis was reached. The study also included enrollment of males, the results of which have been previously reported (16).
Collection of specimens. Following the history and physical examination, clinicians instructed women to collect a 30-ml first-catch urine specimen for LCx LCR (Abbott Laboratories, Abbott Park, Ill.) and Amplicor PCR (Roche Diagnostic Systems, Branchburg, N.J.) tests. At a subsequent pelvic examination, a urethral swab was collected for tissue culture, and endocervical swabs were obtained in the following order: a swab for Gram-stained smear and culture for Neisseria gonorrhoeae; randomly ordered swabs for PACE2 (Gen-Probe), LCR, and PCR; and a swab for tissue culture. The urethral sample was obtained by inserting the swab 2 to 3 cm into the urethral meatus with a rotary motion, and cervical swabs for each nonculture test were obtained according to the manufacturers' instructions. For collection of the endocervical culture specimen, a cytobrush was used according to each center's usual protocol. Urine was held and transported at 4°C.
Laboratory methods. Research laboratories at each center tested urine specimens using the Amplicor microwell plate PCR test and the LCx LCR test. Urine was processed and tests were performed according to the manufacturers' protocols, except that repeated LCR tests for subjects who had equivocal results on initial LCR testing were performed on specimens that had been frozen at –70°C for up to several weeks. Each center used its own protocol for tissue culture isolation of C. trachomatis. Swabs were transported to the laboratory in sucrose phosphate chlamydia transport medium or Eagle's minimal essential medium in Earle salts, each containing fetal calf serum and antibiotics. Specimens were held at 4°C for a maximum of 18 to 72 h, depending upon the center, or were frozen at –70°C. Culture medium was inoculated onto cycloheximide- and/or DEAE-dextran-treated McCoy cells in 96-well microtiter plates or 1-dram vials and incubated at 35 or 37°C for 48 to 72 h. After incubation, the cells were fixed with methanol or ethanol and stained with locally prepared or commercial major outer membrane protein-specific fluorescein-conjugated antibody reagents. One center also used a lipopolysaccharide-specific antibody stain. Three centers performed a blind passage if no chlamydia inclusions were seen at 48 to 72 h. The centers followed the manufacturers' test kit protocols for commercial tests, except that M4 transport medium was used for the PCR cervical test. For N. gonorrhoeae culture, modified Thayer-Martin medium was inoculated at each site and incubated immediately in a CO2-enriched environment at 35 to 37°C for up to 48 h. Testing of urine for the presence of blood and leukocyte esterase was performed by dipstick by laboratory technicians upon receipt of urine samples. For both tests, results were scored as negative, trace, or 1+ to 4+; for analysis, 1+ was considered positive.
Data analysis. As previously reported for these subjects (2), we compared the use of single test reference standards consisting of a culture or NAAT cervical test result with two-specimen reference standards, which consist of cervical culture or cervical NAAT plus urethral culture or urine NAAT results, respectively. A two-specimen reference standard (patient standard), in which subjects are classified as infected if any reference test at any anatomic site is positive, has been advocated to improve reference standard sensitivity and thereby reduce underestimation of test specificity due to false-negative reference standard results (16). The two-specimen standard can be regarded as a patient standard, while the single reference standard may be regarded as a specimen standard. Because a patient standard may be useful for avoiding bias when comparing performances of cervical and urine tests and to maximize the detection of infected patients, we used this approach to define the presence of chlamydial infection. Since neither LCR nor PCR has been shown to be a superior reference standard, we used LCR as the reference standard NAAT when calculating PCR performance and PCR as the reference standard NAAT when calculating LCR performance (2).
The sensitivity and specificity of each test in the presence or absence of key subject characteristics were calculated. These included age, presence of mucopurulent endocervical discharge, report of concurrent menses, concurrent cervical infection with N. gonorrhoeae, and signs and symptoms of urethral inflammation (dysuria and blood or leukocyte esterase in the urine). Because earlier analyses of these data indicated significant differences in test performance across centers for some tests (2), we used a random-effects model for combining proportions to incorporate between-center differences into variance estimates when combining the results from the five centers. Because our results are essentially descriptive, we did not attempt to control for confounding or to analyze interactions with the research center (18). Finally, because an unamplified DNA probe (PACE2) is widely used in clinical practice, we calculated the relative increase in detection of positives by cervical and urine NAAT using cervical PACE2 as the baseline comparison (see Table 3).
RESULTS
Positivity for C. trachomatis. Of 3,551 women enrolled, the plurality were 20 to 24 years old (median age, 24 years) and black and had been seen in STD clinic settings. Table 1 displays C. trachomatis positivity as measured by a patient standard (any single positive test in a cervical, urethral, or urine specimen), unadjusted for differences in test performance across centers (crude positivity). Chlamydia positivity varied significantly across centers, but at all centers it was highest among adolescents. At all centers, positivity was also high among women with mucopurulent endocervical discharge (30.1% as measured by any positive test) and among women with a positive N. gonorrhoeae culture (37%). The results of gonococcal culture were not available for center C. Women who reported contact with a sex partner with C. trachomatis infection had a chlamydia positivity of 44%, while those who reported contact with a partner with N. gonorrhoeae infection had a chlamydia positivity of 29%.
Positivity for C. trachomatis, stratified by test type and key clinical characteristics and adjusted for variations in test performance across centers, is shown in Table 2. Regardless of age, both LCR and PCR detected significantly more positive cervical tests than PACE2 (P < 0.001 to P < 0.03 for separate comparisons), and LCR detected significantly more positive tests at the cervix than did PCR (P 0.001). Regardless of the presence of mucopurulent endocervical discharge, both LCR and PCR detected significantly more positives than PACE2. When mucopurulent endocervical discharge was present, detection of positives by cervical LCR did not differ significantly from that by cervical PCR. However, among women without mucopurulent discharge, while cervical PCR still detected more positives than PACE2, cervical LCR detected more positives than cervical PCR (P < 0.001). LCR and PCR detected similar numbers of positives in the urine of infected women, regardless of age or the presence of mucopurulent endocervical discharge.
Among women who reported concurrent menses, LCR detected more positives at the cervix than either PACE2 or PCR (P < 0.05) (Table 2). Detection of positives was not significantly different, however, for PCR and PACE2 in this group. Of note, for all cervical tests, including NAAT, positivity did not differ significantly in women who reported concurrent menses relative to those who did not. Positivity of NAAT cervical tests exceeded that of NAAT urine tests in women who reported menses; however, this increase was not statistically significant. Among women who did not report concurrent menses, cervical LCR and PCR detected more positives than PACE2 (P < 0.001 and P < 0.02, respectively), while LCR detected more positives than PCR (P < 0.001). Among women with N. gonorrhoeae detected at the cervix, cervical LCR detected significantly more positives than PACE2 (P < 0.001); cervical PCR did not, and LCR detected significantly more positives at the cervix than PCR (P = 0.01). Positivity by either cervical NAAT was higher than by PACE2 among women without cervical gonorrhea (for LCR, P = 0.002; for PCR, P = 0.05).
The relative increase in detection of positives by either NAAT performed at the cervix and in urine over cervical PACE2 is shown in Table 3. In general, use of either cervical or urine LCR was associated with the highest relative increases in estimated positivity, particularly among women who might be expected to have lower organism burdens or a lower expected likelihood of chlamydial infection based on the absence of mucopurulent endocervical discharge or age of >25 years. For example, among women without mucopurulent endocervical discharge, the relative increase in positivity of cervical LCR over PACE2 was high (46%) compared to a 35% relative increase among those with such discharge. Similarly, the relative increase in detection by cervical PCR over PACE2 was higher among women 20 years old than among younger women (31 versus 19%). The highest relative increases in estimated positivity over PACE2 using urine NAAT were seen with urine LCR, particularly among women reporting dysuria (73%). On average, use of the cervical or urine NAAT resulted in an estimated increase of positivity among all subjects of 33% over PACE2.
Calculated performance of tests by cervical and urine findings. Table 4 presents sensitivities, with 95% confidence intervals, of tests performed at the cervix adjusted for variability among test centers as described previously (2, 18). While sensitivities were generally higher in younger women, these differences did not reach statistical significance. Importantly, concurrent menses had no apparent effect on the calculated sensitivity of any individual test. However, PCR sensitivity was significantly higher among women with mucopurulent endocervical discharge than among those without such discharge (P = 0.004). LCR sensitivity was significantly higher among women infected with N. gonorrhoeae than among those not infected (P = 0.05); indeed, in this subgroup of women, we observed the highest sensitivity of any test studied. Interestingly, the presence of hematuria or a positive urine leukocyte esterase test was associated with somewhat higher estimated sensitivities of cervical PCR, but not of any other cervical test.
The estimated specificities of the different cervical tests are summarized in Table 5. Neither concurrent menses nor coinfection with N. gonorrhoeae at the cervix had a significant effect on the calculated specificity of any individual test. However, while the differences did not reach statistical significance, LCR specificity was lower among women with mucopurulent endocervical discharge than among those without such discharge (P = 0.05) and among women infected with N. gonorrhoeae (P = 0.22). PACE2 was less specific among women 20 years old (P = 0.04). The presence of hematuria was associated with higher estimated specificity of cervical LCR, but not of any other cervical test.
The performance of NAAT on urine is summarized in Tables 6 and 7. While both PCR and LCR demonstrated higher sensitivities in the presence of dysuria, mucopurulent endocervical discharge, or hematuria, these differences were not statistically significant. However, the sensitivities of both NAAT were significantly higher among women with a positive urine leukocyte esterase test. The specificities of both tests did not differ by any patient characteristic studied. The sensitivities of NAAT performed on urine differed from those of cervical NAAT only for LCR; cervical LCR was more sensitive than urine LCR among women <20 years old (89 versus 80%, respectively; P = 0.02) and also in women with a negative urine leukocyte esterase test (82 versus 70%; P = 0.01). Urine PCR was less specific than cervical PCR among women who were not menstruating or had no dysuria (99.4 versus 99.7% for both groups; P = 0.04) or who had hematuria (99.3 versus 100%; P = 0.051) or a positive urine leukocyte esterase test (99.2 versus 99.9%; P = 0.03).
DISCUSSION
Among a large number of women attending STD and family planning clinics in the United States, we found that the performances of diagnostic tests for C. trachomatis were selectively affected by some patient characteristics. These effects differed in the NAAT we studied, namely, LCR and the first-generation PCR assay. The important findings were in five areas. First, as we have previously reported for these subjects (2), the LCR assay, whether performed on urine or cervical samples, was generally more sensitive than the other tests studied, even when further analyzed by specific patient characteristics. Second, increased detection by NAAT of cervical C. trachomatis over an unamplified DNA test (PACE2) was most prominent among women without mucopurulent endocervical discharge and among older women (20 years old). Third, the presence of endocervical or urethral inflammation was associated with higher sensitivities of selective NAAT. Specifically, the sensitivity of cervical PCR was significantly higher in the presence of mucopurulent endocervical discharge, and that of cervical LCR was higher in women coinfected with N. gonorrhoeae at the cervix. Similarly, the sensitivities of urine LCR and PCR were higher among women who had a positive urine leukocyte esterase test. Of note, the sensitivity of LCR performed on urine was lower than that of LCR performed at the cervix in adolescent women.
Our fourth finding speaks to previous in vitro data that have suggested that blood may inhibit NAAT (19, 31). We found that concurrent menses had no effect on the performance of either cervical or urine NAAT or on cervical PACE2. It is possible that the increase in cervicovaginal pH that occurs during menses may support a more permissive environment for growth of C. trachomatis (32), perhaps overriding an inhibitory effect of hemoglobin itself. Finally, the effects of subjects' characteristics on the specificities of the tests we studied were less pronounced. For example, cervical LCR demonstrated the lowest specificity in settings associated with its highest sensitivity (for example, women with concurrent endocervical gonococcal infection).
Our finding that urine and cervical NAAT afforded the highest relative increase in detection of C. trachomatis in women who are most likely to have lower burdens of the organism (for example, older women or those without mucopurulent endocervical discharge) is of interest for several reasons. First, it accords with previous findings that NAAT demonstrate the greatest increase in sensitivity over non-NAAT in asymptomatic men without urethral inflammation (22). It also indirectly supports the general concept that the quantity of chlamydial elementary bodies associated with a given infection may correlate directly with signs of inflammation in men and women and with the presence of symptoms in men (12, 22). Second, this observation emphasizes the value of NAAT for screening purposes (defined as testing persons who have no signs or symptoms) (17). However, NAAT remain costly and are unavailable in many settings. Strategies to reduce costs, such as pooling urine samples, may help to facilitate the widespread use of NAAT (17).
Our study has limitations. First, we evaluated the first-generation uniplex PCR assay, modified by replacing the manufacturer's transport medium with a universal transport medium. A modification of this assay in a multiplex format has since replaced the uniplex assay we tested (8, 25). Also, the LCR assay we evaluated is no longer available due to manufacturing issues. However, these problems were not in evidence when we performed our study. Also, we did not evaluate the performance of vaginal-swab specimens, which perform well and are easier to transport than urine (9, 13, 28). While newer NAAT, including strand displacement amplification, transcription-mediated amplification, and more recent iterations of the PCR assay for C. trachomatis, may demonstrate subtle differences by anatomic site and/or by the clinical findings we describe, available data indicate that these assays are very likely to have sensitivities and specificities comparable to those of the LCR assay we studied and superior to those of unamplified DNA probes (3, 30). Second, because our analysis is essentially descriptive, we did not control for potential confounding by research center. We did, however, use statistical methods to incorporate between-center differences into variance estimates when combining the test performance measures from the five centers. We have previously discussed likely reasons for the somewhat lower sensitivities we observed for the urine NAAT in this study relative to values published by others (2). Third, we did not collect data on clinicians' reports of easily induced endocervical bleeding, a common manifestation of mucopurulent cervicitis (MPC) and one criterion for its diagnosis (5). Our analysis thus likely underestimates the true prevalence of MPC among our subjects. Inclusion of women who demonstrated easily induced endocervical bleeding as the sole sign of MPC would likely have increased our estimates for diagnostic test sensitivity and decreased those for specificity in the setting of MPC (20). Finally, most subjects were STD clinic clients, who may not be representative of women tested in other clinical settings. However, we found no differences in test performances between STD clinic clients and women enrolled from the one family planning clinic in our study.
In summary, our findings imply, first, that inflammation, either at the endocervix or the urethra, is associated with a higher organism load and thus confers a higher likelihood of a positive diagnostic test for C. trachomatis. Our observation of higher cervical LCR sensitivity among women with concurrent endocervical gonococcal infection, who may have more inflammation as a result of coinfection with both pathogens, also supports this concept. The second implication of our analysis is that tests with relatively high sensitivity, such as NAAT, exhibit the greatest increase in sensitivity among women with presumably low organism burdens and that reasonably high specificities are maintained in these settings. Our findings support the expanded use of urine (24) and cervical NAAT for both screening and diagnosis of C. trachomatis infection in diverse clinical populations of women. Furthermore, our data emphasize the value these tests bring to comprehensive strategies to reduce the burden of untreated infection in women, who suffer the most costly reproductive tract outcomes of this common sexually transmitted disease.
ACKNOWLEDGMENTS
We thank the staff of the cooperating study sites and the subjects who participated.
This work was supported by the Centers for Disease Control and Prevention, cooperative agreement number 455.
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Division of STD Prevention, National Center for HIV, STD, and TB Prevention
National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Department of Laboratory Medicine, University of California
San Francisco Department of Public Health, San Francisco, California
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
School of Medicine, Indiana University, Indianapolis, Indiana
School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana
ABSTRACT
The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women 20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women.
INTRODUCTION
Genital infection with Chlamydia trachomatis is the most commonly reported bacterial disease in the United States and has well-established adverse effects on the reproductive health of women (4, 23). Nucleic acid amplification tests (NAAT), including PCR, ligase chain reaction (LCR), strand displacement assay, and transcription-mediated amplification, are more sensitive for the diagnosis of C. trachomatis than culture, nonamplified DNA probe, and antigen detection tests and maintain relatively high specificity (1, 2). Equally important, the ability to perform NAAT on urine and self-collected vaginal swabs has expanded opportunities for screening men and women (7, 10, 11, 14, 21, 26, 28, 29).
Our understanding of the clinical manifestations of chlamydial infections in women, including endocervical and urethral inflammation, has been based largely on studies using non-NAAT for detection of infection. Similarly, selective screening criteria for identifying cervical chlamydial infection have been based on risk factors as defined by various non-NAAT. NAAT, however, can detect the presence of fewer chlamydial elementary bodies than non-NAAT (1). Accordingly, among men with urethral chlamydial infection, NAAT demonstrate the greatest increase in sensitivity over non-NAAT in asymptomatic men without urethral inflammation (22). One possible explanation for this is that the quantity of elementary bodies appears to correlate directly with signs of inflammation in men and women and with the presence of symptoms in men (12, 22). Whether the increase in sensitivity of NAAT over non-NAAT is greatest among women with no signs of endocervical or urethral inflammation is not known.
Some data also suggest that NAAT may be inhibited by substances present in clinical specimens, including blood, beta human chorionic gonadotrophin, and products of inflammation (6, 15, 19, 27). However, few studies have directly evaluated whether the performance of NAAT in clinical settings is affected by menstrual blood or by endocervical or urethral inflammation. We recently published an analysis of the performance of NAAT versus an unamplified DNA probe using an expanded and independent diagnostic reference standard in a large, diverse population of women seeking care for family planning or sexually transmitted disease (STD) concerns at five centers across the United States (2). The present study expands these analyses to evaluate the impacts of other factors, including age, endocervical and urethral inflammation, and menstrual blood, on the performance of these tests.
MATERIALS AND METHODS
Women who presented to an STD clinic in each of five participating centers and one family planning clinic who were 16 years old and not pregnant and had not taken antibiotics in the previous 30 days were eligible for enrollment, regardless of genitourinary symptoms. Study clinicians informed eligible patients about the study and enrolled those who gave informed consent. The study was approved by the institutional review board at each center and at the Centers for Disease Control and Prevention. Enrollment began in October 1995 and ended in August 1997, when the target sample of 407 women with urethral or endocervical cultures positive for C. trachomatis was reached. The study also included enrollment of males, the results of which have been previously reported (16).
Collection of specimens. Following the history and physical examination, clinicians instructed women to collect a 30-ml first-catch urine specimen for LCx LCR (Abbott Laboratories, Abbott Park, Ill.) and Amplicor PCR (Roche Diagnostic Systems, Branchburg, N.J.) tests. At a subsequent pelvic examination, a urethral swab was collected for tissue culture, and endocervical swabs were obtained in the following order: a swab for Gram-stained smear and culture for Neisseria gonorrhoeae; randomly ordered swabs for PACE2 (Gen-Probe), LCR, and PCR; and a swab for tissue culture. The urethral sample was obtained by inserting the swab 2 to 3 cm into the urethral meatus with a rotary motion, and cervical swabs for each nonculture test were obtained according to the manufacturers' instructions. For collection of the endocervical culture specimen, a cytobrush was used according to each center's usual protocol. Urine was held and transported at 4°C.
Laboratory methods. Research laboratories at each center tested urine specimens using the Amplicor microwell plate PCR test and the LCx LCR test. Urine was processed and tests were performed according to the manufacturers' protocols, except that repeated LCR tests for subjects who had equivocal results on initial LCR testing were performed on specimens that had been frozen at –70°C for up to several weeks. Each center used its own protocol for tissue culture isolation of C. trachomatis. Swabs were transported to the laboratory in sucrose phosphate chlamydia transport medium or Eagle's minimal essential medium in Earle salts, each containing fetal calf serum and antibiotics. Specimens were held at 4°C for a maximum of 18 to 72 h, depending upon the center, or were frozen at –70°C. Culture medium was inoculated onto cycloheximide- and/or DEAE-dextran-treated McCoy cells in 96-well microtiter plates or 1-dram vials and incubated at 35 or 37°C for 48 to 72 h. After incubation, the cells were fixed with methanol or ethanol and stained with locally prepared or commercial major outer membrane protein-specific fluorescein-conjugated antibody reagents. One center also used a lipopolysaccharide-specific antibody stain. Three centers performed a blind passage if no chlamydia inclusions were seen at 48 to 72 h. The centers followed the manufacturers' test kit protocols for commercial tests, except that M4 transport medium was used for the PCR cervical test. For N. gonorrhoeae culture, modified Thayer-Martin medium was inoculated at each site and incubated immediately in a CO2-enriched environment at 35 to 37°C for up to 48 h. Testing of urine for the presence of blood and leukocyte esterase was performed by dipstick by laboratory technicians upon receipt of urine samples. For both tests, results were scored as negative, trace, or 1+ to 4+; for analysis, 1+ was considered positive.
Data analysis. As previously reported for these subjects (2), we compared the use of single test reference standards consisting of a culture or NAAT cervical test result with two-specimen reference standards, which consist of cervical culture or cervical NAAT plus urethral culture or urine NAAT results, respectively. A two-specimen reference standard (patient standard), in which subjects are classified as infected if any reference test at any anatomic site is positive, has been advocated to improve reference standard sensitivity and thereby reduce underestimation of test specificity due to false-negative reference standard results (16). The two-specimen standard can be regarded as a patient standard, while the single reference standard may be regarded as a specimen standard. Because a patient standard may be useful for avoiding bias when comparing performances of cervical and urine tests and to maximize the detection of infected patients, we used this approach to define the presence of chlamydial infection. Since neither LCR nor PCR has been shown to be a superior reference standard, we used LCR as the reference standard NAAT when calculating PCR performance and PCR as the reference standard NAAT when calculating LCR performance (2).
The sensitivity and specificity of each test in the presence or absence of key subject characteristics were calculated. These included age, presence of mucopurulent endocervical discharge, report of concurrent menses, concurrent cervical infection with N. gonorrhoeae, and signs and symptoms of urethral inflammation (dysuria and blood or leukocyte esterase in the urine). Because earlier analyses of these data indicated significant differences in test performance across centers for some tests (2), we used a random-effects model for combining proportions to incorporate between-center differences into variance estimates when combining the results from the five centers. Because our results are essentially descriptive, we did not attempt to control for confounding or to analyze interactions with the research center (18). Finally, because an unamplified DNA probe (PACE2) is widely used in clinical practice, we calculated the relative increase in detection of positives by cervical and urine NAAT using cervical PACE2 as the baseline comparison (see Table 3).
RESULTS
Positivity for C. trachomatis. Of 3,551 women enrolled, the plurality were 20 to 24 years old (median age, 24 years) and black and had been seen in STD clinic settings. Table 1 displays C. trachomatis positivity as measured by a patient standard (any single positive test in a cervical, urethral, or urine specimen), unadjusted for differences in test performance across centers (crude positivity). Chlamydia positivity varied significantly across centers, but at all centers it was highest among adolescents. At all centers, positivity was also high among women with mucopurulent endocervical discharge (30.1% as measured by any positive test) and among women with a positive N. gonorrhoeae culture (37%). The results of gonococcal culture were not available for center C. Women who reported contact with a sex partner with C. trachomatis infection had a chlamydia positivity of 44%, while those who reported contact with a partner with N. gonorrhoeae infection had a chlamydia positivity of 29%.
Positivity for C. trachomatis, stratified by test type and key clinical characteristics and adjusted for variations in test performance across centers, is shown in Table 2. Regardless of age, both LCR and PCR detected significantly more positive cervical tests than PACE2 (P < 0.001 to P < 0.03 for separate comparisons), and LCR detected significantly more positive tests at the cervix than did PCR (P 0.001). Regardless of the presence of mucopurulent endocervical discharge, both LCR and PCR detected significantly more positives than PACE2. When mucopurulent endocervical discharge was present, detection of positives by cervical LCR did not differ significantly from that by cervical PCR. However, among women without mucopurulent discharge, while cervical PCR still detected more positives than PACE2, cervical LCR detected more positives than cervical PCR (P < 0.001). LCR and PCR detected similar numbers of positives in the urine of infected women, regardless of age or the presence of mucopurulent endocervical discharge.
Among women who reported concurrent menses, LCR detected more positives at the cervix than either PACE2 or PCR (P < 0.05) (Table 2). Detection of positives was not significantly different, however, for PCR and PACE2 in this group. Of note, for all cervical tests, including NAAT, positivity did not differ significantly in women who reported concurrent menses relative to those who did not. Positivity of NAAT cervical tests exceeded that of NAAT urine tests in women who reported menses; however, this increase was not statistically significant. Among women who did not report concurrent menses, cervical LCR and PCR detected more positives than PACE2 (P < 0.001 and P < 0.02, respectively), while LCR detected more positives than PCR (P < 0.001). Among women with N. gonorrhoeae detected at the cervix, cervical LCR detected significantly more positives than PACE2 (P < 0.001); cervical PCR did not, and LCR detected significantly more positives at the cervix than PCR (P = 0.01). Positivity by either cervical NAAT was higher than by PACE2 among women without cervical gonorrhea (for LCR, P = 0.002; for PCR, P = 0.05).
The relative increase in detection of positives by either NAAT performed at the cervix and in urine over cervical PACE2 is shown in Table 3. In general, use of either cervical or urine LCR was associated with the highest relative increases in estimated positivity, particularly among women who might be expected to have lower organism burdens or a lower expected likelihood of chlamydial infection based on the absence of mucopurulent endocervical discharge or age of >25 years. For example, among women without mucopurulent endocervical discharge, the relative increase in positivity of cervical LCR over PACE2 was high (46%) compared to a 35% relative increase among those with such discharge. Similarly, the relative increase in detection by cervical PCR over PACE2 was higher among women 20 years old than among younger women (31 versus 19%). The highest relative increases in estimated positivity over PACE2 using urine NAAT were seen with urine LCR, particularly among women reporting dysuria (73%). On average, use of the cervical or urine NAAT resulted in an estimated increase of positivity among all subjects of 33% over PACE2.
Calculated performance of tests by cervical and urine findings. Table 4 presents sensitivities, with 95% confidence intervals, of tests performed at the cervix adjusted for variability among test centers as described previously (2, 18). While sensitivities were generally higher in younger women, these differences did not reach statistical significance. Importantly, concurrent menses had no apparent effect on the calculated sensitivity of any individual test. However, PCR sensitivity was significantly higher among women with mucopurulent endocervical discharge than among those without such discharge (P = 0.004). LCR sensitivity was significantly higher among women infected with N. gonorrhoeae than among those not infected (P = 0.05); indeed, in this subgroup of women, we observed the highest sensitivity of any test studied. Interestingly, the presence of hematuria or a positive urine leukocyte esterase test was associated with somewhat higher estimated sensitivities of cervical PCR, but not of any other cervical test.
The estimated specificities of the different cervical tests are summarized in Table 5. Neither concurrent menses nor coinfection with N. gonorrhoeae at the cervix had a significant effect on the calculated specificity of any individual test. However, while the differences did not reach statistical significance, LCR specificity was lower among women with mucopurulent endocervical discharge than among those without such discharge (P = 0.05) and among women infected with N. gonorrhoeae (P = 0.22). PACE2 was less specific among women 20 years old (P = 0.04). The presence of hematuria was associated with higher estimated specificity of cervical LCR, but not of any other cervical test.
The performance of NAAT on urine is summarized in Tables 6 and 7. While both PCR and LCR demonstrated higher sensitivities in the presence of dysuria, mucopurulent endocervical discharge, or hematuria, these differences were not statistically significant. However, the sensitivities of both NAAT were significantly higher among women with a positive urine leukocyte esterase test. The specificities of both tests did not differ by any patient characteristic studied. The sensitivities of NAAT performed on urine differed from those of cervical NAAT only for LCR; cervical LCR was more sensitive than urine LCR among women <20 years old (89 versus 80%, respectively; P = 0.02) and also in women with a negative urine leukocyte esterase test (82 versus 70%; P = 0.01). Urine PCR was less specific than cervical PCR among women who were not menstruating or had no dysuria (99.4 versus 99.7% for both groups; P = 0.04) or who had hematuria (99.3 versus 100%; P = 0.051) or a positive urine leukocyte esterase test (99.2 versus 99.9%; P = 0.03).
DISCUSSION
Among a large number of women attending STD and family planning clinics in the United States, we found that the performances of diagnostic tests for C. trachomatis were selectively affected by some patient characteristics. These effects differed in the NAAT we studied, namely, LCR and the first-generation PCR assay. The important findings were in five areas. First, as we have previously reported for these subjects (2), the LCR assay, whether performed on urine or cervical samples, was generally more sensitive than the other tests studied, even when further analyzed by specific patient characteristics. Second, increased detection by NAAT of cervical C. trachomatis over an unamplified DNA test (PACE2) was most prominent among women without mucopurulent endocervical discharge and among older women (20 years old). Third, the presence of endocervical or urethral inflammation was associated with higher sensitivities of selective NAAT. Specifically, the sensitivity of cervical PCR was significantly higher in the presence of mucopurulent endocervical discharge, and that of cervical LCR was higher in women coinfected with N. gonorrhoeae at the cervix. Similarly, the sensitivities of urine LCR and PCR were higher among women who had a positive urine leukocyte esterase test. Of note, the sensitivity of LCR performed on urine was lower than that of LCR performed at the cervix in adolescent women.
Our fourth finding speaks to previous in vitro data that have suggested that blood may inhibit NAAT (19, 31). We found that concurrent menses had no effect on the performance of either cervical or urine NAAT or on cervical PACE2. It is possible that the increase in cervicovaginal pH that occurs during menses may support a more permissive environment for growth of C. trachomatis (32), perhaps overriding an inhibitory effect of hemoglobin itself. Finally, the effects of subjects' characteristics on the specificities of the tests we studied were less pronounced. For example, cervical LCR demonstrated the lowest specificity in settings associated with its highest sensitivity (for example, women with concurrent endocervical gonococcal infection).
Our finding that urine and cervical NAAT afforded the highest relative increase in detection of C. trachomatis in women who are most likely to have lower burdens of the organism (for example, older women or those without mucopurulent endocervical discharge) is of interest for several reasons. First, it accords with previous findings that NAAT demonstrate the greatest increase in sensitivity over non-NAAT in asymptomatic men without urethral inflammation (22). It also indirectly supports the general concept that the quantity of chlamydial elementary bodies associated with a given infection may correlate directly with signs of inflammation in men and women and with the presence of symptoms in men (12, 22). Second, this observation emphasizes the value of NAAT for screening purposes (defined as testing persons who have no signs or symptoms) (17). However, NAAT remain costly and are unavailable in many settings. Strategies to reduce costs, such as pooling urine samples, may help to facilitate the widespread use of NAAT (17).
Our study has limitations. First, we evaluated the first-generation uniplex PCR assay, modified by replacing the manufacturer's transport medium with a universal transport medium. A modification of this assay in a multiplex format has since replaced the uniplex assay we tested (8, 25). Also, the LCR assay we evaluated is no longer available due to manufacturing issues. However, these problems were not in evidence when we performed our study. Also, we did not evaluate the performance of vaginal-swab specimens, which perform well and are easier to transport than urine (9, 13, 28). While newer NAAT, including strand displacement amplification, transcription-mediated amplification, and more recent iterations of the PCR assay for C. trachomatis, may demonstrate subtle differences by anatomic site and/or by the clinical findings we describe, available data indicate that these assays are very likely to have sensitivities and specificities comparable to those of the LCR assay we studied and superior to those of unamplified DNA probes (3, 30). Second, because our analysis is essentially descriptive, we did not control for potential confounding by research center. We did, however, use statistical methods to incorporate between-center differences into variance estimates when combining the test performance measures from the five centers. We have previously discussed likely reasons for the somewhat lower sensitivities we observed for the urine NAAT in this study relative to values published by others (2). Third, we did not collect data on clinicians' reports of easily induced endocervical bleeding, a common manifestation of mucopurulent cervicitis (MPC) and one criterion for its diagnosis (5). Our analysis thus likely underestimates the true prevalence of MPC among our subjects. Inclusion of women who demonstrated easily induced endocervical bleeding as the sole sign of MPC would likely have increased our estimates for diagnostic test sensitivity and decreased those for specificity in the setting of MPC (20). Finally, most subjects were STD clinic clients, who may not be representative of women tested in other clinical settings. However, we found no differences in test performances between STD clinic clients and women enrolled from the one family planning clinic in our study.
In summary, our findings imply, first, that inflammation, either at the endocervix or the urethra, is associated with a higher organism load and thus confers a higher likelihood of a positive diagnostic test for C. trachomatis. Our observation of higher cervical LCR sensitivity among women with concurrent endocervical gonococcal infection, who may have more inflammation as a result of coinfection with both pathogens, also supports this concept. The second implication of our analysis is that tests with relatively high sensitivity, such as NAAT, exhibit the greatest increase in sensitivity among women with presumably low organism burdens and that reasonably high specificities are maintained in these settings. Our findings support the expanded use of urine (24) and cervical NAAT for both screening and diagnosis of C. trachomatis infection in diverse clinical populations of women. Furthermore, our data emphasize the value these tests bring to comprehensive strategies to reduce the burden of untreated infection in women, who suffer the most costly reproductive tract outcomes of this common sexually transmitted disease.
ACKNOWLEDGMENTS
We thank the staff of the cooperating study sites and the subjects who participated.
This work was supported by the Centers for Disease Control and Prevention, cooperative agreement number 455.
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