Over the course of the last two years, Clinical Pathology Laboratories’ (CPL) main laboratory in Austin, Texas was involved in two large-scale transitions involving syphilis confirmatory testing. In 2018, our lab transitioned from the manual rapid plasma reagin flocculation test to a rapid plasma reagin (RPR) assay-based automated test system, the AIX1000 RPR from Gold Standard Diagnostics. This move was largely precipitated by a desire to obviate the manual requirements of the card flocculation test coupled with the goal of meeting our expected turnaround time (TAT) metrics given a test volume of greater than 30,000 tests per month.
While the direct material costs were expected to increase by almost 50% as a result of this transition, the hematology department forecasted a significant direct labor savings, which translated to a projected ROI of less than five years. The anticipated quality improvements resulting from the standardization of specimen processing and image analysis were deemed additional benefits of this new technology.
Laboratory Methodology Transitions
Simply, the automated test system delivers serum to microtiter plate wells, an antigen mixture is added, and plates are incubated while shaking. A camera captures the image and proprietary software employs an algorithm to determine the results. Reactive specimens are then diluted with PBS buffer to determine an endpoint titer up to a ratio of 1:256. Upon validation, it was anticipated that there would be subtly increased sensitivity with decreased specificity.
In 2019, CPL brought in house testing for T. Pallidum-particle agglutination (TP-PA), a manual process that was previously sent to an outside referral laboratory, which confirmed both MHA-TP and FTA-ABS testing. While this decision was somewhat counter culture to our general initiative toward automated testing, this transition represented an opportunity to reduce costs through the absorption of referral testing, improve client retention, and offer greater ownership in the continuum of care for clients requesting this test.
For TP-PA, serum samples are now diluted in microtiter plate wells to a ratio of 1:20 or 1:40 and 25 μl of unsensitized or sensitized gelatin particles is added to the respective wells. Unsensitized gelatin particles help determine if the patient’s immune response is to the antigen on the particle surface or the particle itself. Plates are shaken for 30 seconds and then incubated under stationary conditions for 2 hours at room temperature.
The clinical manifestations of syphilis—a chronic, sexually transmitted disease caused by the spirochete, Treponema pallidum (the causative organism)—are often non-specific and progressive if not treated promptly.1 Resurgences in various geographic areas have required more stringent analysis to ensure efficient use of resources. Quebec, for example, only reported three cases of syphilis in 1998, but that number rose to a rate of 7.1 per 100,000 people in 2015.2
The diagnosis of syphilis infection has historically relied upon both treponemal and nontreponemal (NTT) methodologies but was limited by the lack of a widely accepted gold standard diagnostic test. Treponemal tests use specific antibodies to T. pallidum while NTT tests detect antibodies formed in response to cellular damage.3 The interpretations of these tests are further subdivided with their individual modes of interpretative guidance.
The sensitivity of the RPR assay ranges from 73% in latent syphilis to 100% in secondary syphilis, though the time associated with the highest likelihood of transmission of the infection, primary syphilis, represents a sensitivity of 86%.4,5
A number of test platforms are available on the market for initial NTT RPR screening. The manual flocculation-based Becton Dickinson Macro-Vue test kit is widely regarded as the most cost-effective. More automated methods exist in the form of the Arlington Scientific ASI Evolution and Gold Standard Diagnostics AIX1000. Either of these automated platforms provide high-throughput standardized methods that stores results in a central database.
Evolving Screening Recommendations
The traditional screening algorithm pathway (see FIGURE 1) relies on the initial analysis of a NTT such as the RPR (rapid plasma reagin) or Venereal Disease Research Laboratory (VDRL) test. If the initial result is reactive, then additional testing is done to confirm the primary result. This additional testing can be done a number of ways including via TP-PA, fluorescent treponemal antibody absorption (FTA-ABS), enzyme immunoassay (EIA), or chemiluminescence immunoassay (CLIA).
A reactive result on this confirmatory test is to be considered diagnostic of active T. pallidum infection. Additional specificity for T. pallidum diagnosis is achieved with IgG- and/or IgM-class antibodies detected with the NTT methods described above.
The reverse algorithm (see FIGURE 2) is another alternative used by health care providers. Recent studies have indicated that reverse sequence screening may detect more cases of early or latent syphilis than the traditional forward screening algorithm.6 As recommended by the Centers for Disease Control and Prevention (CDC), discordant syphilis IgG and RPR results are to be resolved by a second treponemal test (TP-PA).7 In the presence of a positive syphilis IgG and non-reactive RPR, a non-reactive treponemal test indicates a false positive syphilis IgG screen because TP-PA has a higher sensitivity than syphilis IgG screening.8 A treponemal confirmation test deemed positive typically results in patient treatment, as the regimen is inexpensive and the consequences of a missed public health opportunity can be large.
This reverse algorithm pathway is gaining traction among obstetric health care providers due to the cross-reactivity of the RPR test with medical conditions such as other infections (eg, HIV), pregnancy, autoimmune disorders, injection-drug use, older age, or immunizations. Screening out this approximately 1% of the population that exists as false positive has become a necessity (see FIGURE 3). Aside from the obvious benefits of lab automation, this method also represents less intensive patient follow-up and stress from treatment. Although not as widely preferred as the forward algorithm, CPL provides guidance to clinicians for interpreting reverse algorithm results (see FIGURE 4). Both of these approaches are summarized in FIGURE 5.
One significant potential limitation of using the reverse algorithm exclusively is the prevalence of the impact of false-positive results that can contribute to a burden of anxiety and treatment.9 However, other more obvious benefits of the reverse screening pathway include the ability to perform the treponemal IgG test on a high throughput analyzer (such as the Bio-Rad BioPlex 2200). Furthermore, the quantitative result obtained from this test aids in the monitoring of disease progression, especially during the tertiary phase. The reverse screening process studied by the University of Iowa Hospitals and Clinics has proven that 99% of results screened negative (1% screened positive).10 While the initial IgG treponemal test may be more expensive per test, overall client satisfaction is improved by providing results more rapidly.
Part 2 of this article will discuss the automation initiative, analytical process monitoring, and overall management considerations.
Special thanks to Mr. Michael Callaway, Director of Technical Operations, Dr. Mark Silberman, Laboratory Director, Dr. Kim Monnin, Hematology Medical Director, and Dr. Christine Burgess, Chemistry Medical Director, Clinical Pathology Laboratories, Austin, TX.
Aaron Samson, MBA, MB(ASCP)CM, is the hematology operations manager at CPL in Austin, Texas, where he oversees staff in various areas of specimen testing. Prior to joining CPL in 2012, Aaron performed high complexity flow cytometry and molecular testing at the University of Kentucky Medical Center. He has experience with lab automation implementations, project management, and process improvement.
Enter our Sweepstakes now for your chance to win the following prizes:
Just answer the following quick question for your chance to win:
Entries are limited to one entry per person in any active sweepstakes.
Thank you for your entry.