A 1-year evaluation of Syva MicroTrak Chlamydia enzyme immunoassay with selective confirmation by direct fluorescent-antibody assay in a high-volume laboratory

Abstract

The Syva MicroTrak Chlamydia enzyme immunoassay (EIA; Syva Company, San Jose, Calif.) with cytospin and direct fluorescent-antibody assay (DFA) confirmation was evaluated on 43,630 urogenital specimens over a 1-year period in the Provincial Laboratory in Regina, Saskatchewan, Canada. This was a two-phase study intended to define a testing algorithm for Chlamydia trachomatis that would be both highly accurate and cost-effective in our high-volume (>3,000 tests per month) laboratory. The prevalence of C. trachomatis infection in our population is moderate (8 to 9%). In phase 1, we tested 6,022 male and female urogenital specimens by EIA. All specimens with optical densities above the cutoff value and those within 30% below the cutoff value were retested by DFA. This was 648 specimens (10.8% of the total). A total of 100% (211 of 211) of the specimens with optical densities equal to or greater than 1.00 absorbance unit (AU) above the cutoff value, 98.2% (175 of 178) of the specimens with optical densities of between 0.500 and 0.999 AU above the cutoff value, and 83% (167 of 201) of the specimens with optical densities within 0.499 AU above the cutoff value were confirmed to be positive. A total of 12% (7 of 58) of the specimens with optical densities within 30% below the cutoff value were positive by DFA. In phase 2, we tested 37,608 specimens (32,495 from females; 5,113 from males) by ETA. Only those specimens with optical densities of between 0.499 AU above and 30%o below the cutoff value required confirmation on the basis of data from phase 1 of the study. This was 4.5% of all specimens tested. This decrease in the proportion of specimens requiring confirmation provides a significant cost savings to the laboratory. The testing algorithm gives us a 1-day turnaround time to the final confirmed test results.The MicroTrak ETA performed very well in both phases of the study, with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.1, 99.1, 90.3, and 99.7%, respectively, in phase 2. We suggest that for laboratories that use ETA for Chlamydia testing, a study such as this one will identify an appropriate optical density range for confirmatory testing for samples from that particular population.

Current Methods of Laboratory Diagnosis of Chlamydia Trachomatis Infections

“…Although the DFA test is still used in many laboratories as a primary diagnostic test for C. trachomatis, the labor and skill required to perform the test limit its use to low volumes of specimens. As it has lost favor as a primary diagnostic test, the DFA test has become popular as a confirmation test for positive results of other nonculture C. trachomatis tests (33,98) and in discrepancy analysis schemes for DNA amplification tests because of its high specificity (Tables 6 to 8). In confirmation and discrepancy analyses, the cutoff for a positive test is generally 2 elementary bodies.…”

“…In confirmation and discrepancy analyses, the cutoff for a positive test is generally 2 elementary bodies. Protocols for performing DFA tests as confirmatory tests vary but generally consist of centrifugation of the original specimen in transport buffer onto a microscope slide followed by fixation and staining by the conventional DFA method (33,98). This procedure has been most often used to confirm the results of EIA for antigen detection and to assess the adequacy of the specimens for EIA, but it has also been used to examine sediments of centrifuged urine (111,202).…”

“…Blocking antibody reagent should be used only with specimens with which it has been validated. An alternative confirmation procedure for EIA is to perform a DFA test on the centrifuged specimen (14,33,148).…”

“…The sensitivity of Chlamydiazyme improves with the use of either DFA or blocking antibody for confirmation of "gray-zone" specimens below the absorbance cutoff (98). The use of confirmation with blocking antibody reagent has been reported in several studies to reduce the proportion of false-positive results with Chlamydiazyme (33,39,94) and is mandatory when testing urine specimens because of the presence of contaminating gram-negative bacteria (6,50). The blocking antibody reagent used for Chlamydiazyme has been found in one study to agree well with confirmation by DFA tests with MOMP-specific monoclonal antibody (67), but in another study, 24% of EIA-reactive, blocking test-negative specimens were positive by DFA (244).…”

“…Several studies have shown that without quality assurance of specimen adequacy, more than 10% of specimens will be unsatisfactory because they contain exudate and lack urethral or endocervical columnar cells (95,96,118). Two methods have been developed for the determination of specimen adequacy by visualization of columnar cells: (i) performing a direct specimen smear, staining with a chlamydia-specific fluorescent antibody followed by a counterstain or by Giemsa stain, and (ii) centrifugation of an aliquot of a specimen in non-detergent-based transport medium, such as that used for culture or EIA, onto a microscope slide and staining by a DFA test (33,63). For determination of specimen adequacy, many laboratorians find a rapid differential hematology stain such as Diff-Quik (Baxter Diagnostics, McGaw Park, Ill.) easier to read than fluorescent antibody-stained slides due to improved differentiation of cell types.…”