Use of Multiple Nucleic Acid Amplification Tests To Define the Infected-Patient “Gold Standard” in Clinical Trials of New Diagnostic Tests for Chlamydia trachomatis Infections

Abstract

Nucleic acid amplification tests (NAATs) can be used to define the infected-patient "gold standard" for the purpose of designing studies of the performance of Chlamydia trachomatis diagnostic tests. It is unclear how many test results run by different NAATs and what combinations of specimens comprise the best infectedpatient gold standard. We approached this question with data from a large study of the performance of a new NAAT. Data were available from three endocervical swabs and a urine specimen collected from each of 1,412 women and tested by three different NAATs. Results from all three assays were used equally in a rotating fashion to define the infected-patient gold standard. Multiple different infected-patient gold standards for estimating swab and urine specimen sensitivity and specificity for one NAAT method were created by varying the number and combinations of swab and urine comparator results with two different NAATs, The effect of changing the infected-patient gold standard definition was determined by constructing receiver-operator-like curves with calculated sensitivities and specificities for each test. The one-positive-of-two-results or twopositive-of-two-results (same or two different assays) infected-patient gold standard definitions produced low sensitivity and low specificity estimates, respectively. If four comparator NAAT results were used, the anythree-positive-of-four-results definition or the at-least-one-specimen-positive-by-each-of-two-comparator-assays definition appeared to provide better combinations of sensitivity and specificity estimates. The any-twopositive-out-of-three-results definition resulted in estimates that were as good as produced with the former two definitions. This analytic approach provides a means of clearly visualizing the effects of changing NAAT-based infected-patient gold standards and should be helpful in designing future studies of new C. trachomatis diagnostic tests.Determination of the infected patient "gold standard" for measuring the performance of new nucleic acid amplification tests (NAATs) for the detection of Chlamydia trachomatis in genitourinary specimens has proven difficult. Historically, culture was the gold standard for determining the performance characteristics of new diagnostic tests for this organism. Based on the analytical sensitivity of NAATs in addition to the fact that it was known that culture was not optimally sensitive, it was reasonably clear initially that these assays would be more sensitive than C. trachomatis culture. Indeed, studies showed that the use of culture alone as a reference standard resulted in significant underestimates of the specificity for NAATs as many infected patients were considered falsely negative by culture (7, 13).To address this problem, investigators applied an alternative target amplification assay to the putative false-positive results (discrepancy analysis). There was no alternative at the time, as tests with similar sensitivity were not available for use in a composite infected-patient definition a...

Comparative performance of the Roche COBAS Amplicor assay and an in-house real-time PCR assay for diagnosis of Chlamydia trachomatis infection

“…However, a recent publication has cast doubt about the validity of this approach due to the poor sensitivity of culture compared with NAATs (Jespersen et al, 2005). The use of multiple molecular assays provides an appropriate reference for investigating the performance of new NAATs (Martin et al, 2004). This principle was used to define the new gold standard used for the present study.…”

High Analytical Sensitivity and Low Rates of Inhibition May Contribute to Detection of Chlamydia trachomatis in Significantly More Women by the APTIMA Combo 2 Assay

“…These are significant and clinically important differences not observed in previous published studies, which may be explained by several factors. Most clinical comparisons have used two or three different assays on one or two specimen types (3,8,(13)(14)(15)(26)(27)(28)(29). Our study had the unique design of collecting three VS, three CS, and FVU from each patient and enabled us to expand the reference standard.…”

“…This type of study maneuver usually lowers the sensitivity of single specimen evaluations (13,14). We elected to declare a patient infected if two or more samples were positive in at least one of the assays or if at least one sample was positive in more than one assay.…”

Nucleic Acid Amplification Tests for Diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis Rectal Infections

“…Participants with negative test sets may have had more than one test set included in the analysis if the subsequent swab collection took place 30 days or more following the initial collection. Test sensitivity and specificity were calculated using a rotating standard which compared each test under evaluation with a performance standard that classified subjects as infected if two or more of the three remaining comparator tests were positive (2,12). A second standard was applied in which each test was compared to a performance standard that classified subjects as infected only if all three of the remaining comparator tests were positive.…”