点击显示 收起
Magee-Womens Research Institute,1 Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania2
Received 31 January 2003/ Returned for modification 31 March 2003/ Accepted 6 May 2003
ABSTRACT |
---|
Top Abstract Introduction Materials and Methods Results Discussion References |
---|
INTRODUCTION |
---|
Top Abstract Introduction Materials and Methods Results Discussion References |
---|
The increased sensitivities of nucleic acid amplification techniques have led to the evaluation of less invasive procedures for screening for sexually transmitted pathogens. The vaginal introitus (17) and vulva (12) have been shown to be acceptable sites for noninvasive sampling with swabs for the detection of C. trachomatis. It has been shown that patient-obtained vaginal swab specimens can be used to accurately detect N. gonorrhoeae infections (4). Noninvasive collection methods allow women to be screened without the need for a speculum examination. Vulvar swabbing (13) and vaginal flushing (8) have been used to collect specimens that were mailed to a laboratory. The self-collection of vaginal swab specimens at home may be more acceptable for the screening of young people who have limited contact with health services (8, 13).
SDA has already been shown to be an acceptable method for the detection of C. trachomatis and N. gonorrhoeae with urine and endocervical swab specimens (14). The purpose of the present study was to compare vaginal swab specimens to endocervical swab specimens for the detection of C. trachomatis and N. gonorrhoeae. The swabs used for SDA are transported medium-free and are therefore less prone to spillage during transport than urine samples. A second goal of this study was to evaluate the utility of the amplification control for vaginal swab specimens in a clinical research setting.
(This study was presented in part at the 101st General Meeting of the American Society for Microbiology, Orlando, Fla., 20 to 24 May 2001.)
MATERIALS AND METHODS |
---|
Top Abstract Introduction Materials and Methods Results Discussion Reerences |
---|
In the implementation phase of the study, vaginal swab specimens were obtained consecutively from 2,973 women at five different geographic locations. Written informed consent approved by each institutional review board was obtained from all participants prior to the initiation of study procedures. Clinician- or patient-obtained vaginal swab specimens for SDA were collected by using the Culturette Direct specimen collection and dry transport kit (Becton Dickinson), delivered to the laboratory, and processed within 6 days according to the guidelines of the manufacturer.
For SDA, the swab contents were expressed into tubes containing 2 ml of sample diluent. The tube was then placed into a lysing rack and heated at 114°C for 30 min. The samples were then removed from the heater and cooled for 15 min at room temperature. The samples were transferred to the priming microwells and incubated at room temperature for 20 min. The priming microwell plate and the amplification microwell plate were then placed in the priming heater for 10 min at 72.5 and 54°C, respectively. At the end of the 10 min, 100 µl was transferred from the priming microwells to the amplification microwells. The amplification microwell plate was then sealed with an amplification sealer and placed in the BDProbeTec ET instrument. The run was initiated for 60 min at 52.5°C. The presence or absence of C. trachomatis and N. gonorrhoeae was determined by relating the BDProbeTec ET method-other-than-acceleration scores for the specimen to predetermined cutoff values (7).
Amplicor PCR (Roche Diagnostic Systems) analysis was performed according to the instructions in the manufacturer's package insert. PCR amplification was conducted with a Thermocycler TC 9600 instrument (Perkin-Elmer Cetus, Norwalk, Conn.). After the amplification, the amplified nucleotide sequences were detected by using target-specific DNA probes. The resulting enzyme reaction was measured with a spectrophotometer (Molecular Devices, Sunnyvale, Calif.). Specimens with an A450 of 0.500 were considered positive (1).
The endocervical swab specimen for the culture detection of N. gonorrhoeae was stored at ambient temperature and inoculated onto modified Thayer-Martin medium and chocolate agar (PML Microbiologicals, Tualatin, Oreg.) within 24 h of collection. The plates were placed in 5 to 7% CO2 at 36°C for up to 48 h (6), after which they were examined for the presence of N. gonorrhoeae. Identification was based on Gram staining, the oxidase test, and the Gonochek II identification system (EY Laboratories, Inc., San Mateo, Calif.).
In the case of a disagreement between the results of SDA and PCR or the results of SDA and culture, samples with discrepant results were evaluated by the LCR assay. The samples to be tested by LCR were stored at -20°C until they were transferred to the Clinical Microbiology Laboratory at Magee-Womens Hospital. The LCR assay was performed according to the instructions in the manufacturer's package insert (1).
For C. trachomatis, a sample was considered to have a true-positive result if it was positive by two molecular tests (PCR and SDA, PCR and LCR, or LCR and SDA). For N. gonorrhoeae, a sample was considered to have a true-positive result if it was positive by culture or positive by two molecular tests (SDA and LCR).
RESULTS |
---|
Top Abstract Introduction Materials and Methods Results Discussion References |
---|
|
Indeterminate assay results with vaginal swab specimens continued to present a challenge with respect to having to recall patients for retesting. Therefore, another series of vaginal swab samples was concurrently evaluated by using a 1:1 dilution of the specimen and an undiluted specimen. Of the 236 specimens evaluated in this manner, 56 (23.7%) were found to have indeterminate results for one or both analytes. Overall, when the results for the undiluted specimens were compared to those for the specimens diluted 1:1, 49 of the 56 specimens (88%) with indeterminate results were negative. The remaining seven samples, which had indeterminate results for C. trachomatis by SDA, were tested by PCR, and six were confirmed to be negative for C. trachomatis and one sample was also found to inhibit the PCR assay. Among the 236 specimens evaluated in this series, routine 1:1 dilution yielded 4 specimens newly positive for N. gonorrhoeae, but the results for 2 specimens initially positive for C. trachomatis became indeterminate with repeat testing of the sample diluted 1:1, and 1 specimen initially positive for N. gonorrhoeae became negative. This series of evaluations revealed that retesting of samples indeterminate by SDA by repeating SDA or PCR usually yielded negative results. To confirm this, an additional 1,411 vaginal swab specimens were evaluated by SDA with the amplification control. All specimens whose results were indeterminate by SDA and for which the results were not resolved by repeat testing were evaluated by PCR. Of 357 specimens with indeterminate results by SDA, the results for 316 (89%) were resolved after repeat testing by SDA. The remaining 41 specimens were retested by PCR; and 36 specimens were negative for C. trachomatis, 3 specimens were inhibitory, and 2 specimens yielded equivocal results. Of the 41 vaginal swab samples tested by PCR, only 7% inhibited the PCR, even though all specimens inhibited SDA, suggesting that the two assays are inhibited by different factors.
In summary, a total of 1,647 vaginal swab specimens were tested for C. trachomatis and N. gonorrhoeae by SDA. Of the undiluted vaginal swab samples, 413 (25%) yielded indeterminate test results and required further testing. Following 1:1 dilution of the specimen and repeat testing, the prevalences of C. trachomatis and N. gonorrhoeae in the group of 1,647 specimens were 6.0 and 3.1%, respectively (Table 2). However, even after dilution and repeat testing, only 1.5% of the specimens that initially had indeterminate results and whose results were resolved were found to be positive. If the original samples had been evaluated without the amplification control and all of the indeterminate results had been interpreted as negative, the prevalence rate for C. trachomatis would have been 5.8% (95 of 1,647) and that of N. gonorrhoeae would have been 3.0% (49 of 1,647). Thus, the repeat testing and resolution of the results by PCR for samples with indeterminate results had a limited overall impact on the prevalence of positivity in this population.
|
DISCUSSION |
---|
Top Abstract Introduction Materials and Methods Results Discussion References |
---|
The amplified DNA assays with the BDProbeTec ET instrument were designed so that endocervical swab, urethral swab, or urine specimens could be evaluated for the detection of C. trachomatis and N. gonorrhoeae. However, other studies of amplified tests for the detection of these organisms have shown that vaginal swab specimens are an acceptable specimen type. For example, tests with vaginal swab specimens were found to be 100% sensitive and 99.6% specific for the detection of N. gonorrhoeae by LCR (4), while tests with vaginal swab specimens were reported to be 92% sensitive and 100% specific for the detection of C. trachomatis by PCR (17). The manufacturer's instructions for the collection of cervical swab specimens include the removal of excess mucus from the cervix with a large-tipped cleaning swab before insertion of the collection swab into the cervical os. In contrast, the vagina is a moist epithelium that does not secrete mucus. Nevertheless, cervical mucus is present in the vaginal fluid and may be present in the vaginal swab specimen, leading to the potential for sample inhibition by mucus or other vaginal fluid components.
Use of the amplification control during testing is an option. The amplification control is designed to identify samples that may contain inhibitors that could prevent the detection of C. trachomatis or N. gonorrhoeae. However, when test results are interpreted, a positive result may be interpreted for any amplification control value. An indeterminate result is interpreted only for samples with low scores for C. trachomatis or N. gonorrhoeae score, i.e., <2,000, which is also the lower limit for positive results. The present study suggests that most vaginal swab specimens with indeterminate results by SDA have true-negative results.
During the evaluation phase of the study, the rate of indeterminate results was 0.7% in tests with cervical swab specimens and 6.4% in tests with vaginal swab specimens. This phase of the study was conducted at a single site, and only three clinicians collected the specimens. However, in the research implementation phase of the study, about 25% of the vaginal swab specimens gave indeterminate results. It is unknown why the rate of indeterminate results increased. However, during the implementation phase of the study, five different clinical sites were involved, and a large number of different people collected samples. A first strategy to resolve indeterminate results was to repeat the test with undiluted specimens. However, repeat testing in this fashion resolved the results for only a third of the samples. Indeterminate results by the repeat test led to clinician notification that the patient should be recalled for collection and testing of another specimen. The need to recall patients for repeat testing was costly and caused the patients concern.
Our goal was to determine whether samples could be diluted routinely to reduce the number of samples with indeterminate results without the expense of retesting or a loss of test sensitivity. We found that routine dilution of the specimens resolved the indeterminate results, but at the cost of decreased sensitivity. Repeat testing was also very costly. The cost for the testing of the 2,973 samples evaluated during the second phase of this study and repeat testing to resolve the results for samples with indeterminate results was $13,000 for supplies alone. Tests for the detection of six additional positive specimens cost $2,166 in additional supplies per infection detected. By excluding use of the amplification control, there is a minimal difference in supply costs ($0.11/sample) and there is no basis for repeat testing. Data included in the BDProbeTec ET package insert note that use of the amplification control increased the sensitivity of the C. trachomatis test from 92.0 to 92.8% and decreased the specificity slightly, from 96.6 to 96.1%. When the amplification control was used for the testing of N. gonorrhoeae, the sensitivity increased from 96.4 to 97.6% and the specificity remained 99%. Thus, our findings with vaginal swab specimens are similar to those cited by the manufacturer in the package insert, in that the use of the amplification control had a minimal impact on test performance. The Food and Drug Administration does not require the use of the amplification control for the evaluation of cervical and urine specimens, and our data collected in a clinical research setting suggest that this control has limited value when vaginal swab specimens are tested.
From this study, we have concluded that vaginal swab specimens are an acceptable alternative to cervical swab specimens for the detection of N. gonorrhoeae and C. trachomatis by SDA with the BDProbeTec ET system. Furthermore, we have determined that use of the amplification control with this specimen type increases costs and can result in delay in test reporting due to the high frequency of indeterminate results.
ACKNOWLEDGMENTS |
---|
This study was supported in part by a grant (grant DAMD 17-96-1-6298) from the U.S. Department of Defense.
REFERENCES |
---|
Top Abstract Introduction Materials and Methods Results Discussion References |
---|