CAP Laboratory Improvement Programs. Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice
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1 CAP Laboratory Improvement Programs Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice A Survey of Participants in the College of American Pathologists Syphilis Serology Proficiency Testing Program Daniel D. Rhoads, MD; Jonathan R. Genzen, MD, PhD; Christine P. Bashleben, MT(ASCP); James D. Faix, MD; M. Qasim Ansari, MD Context. Syphilis serology screening in laboratory practice is evolving. Traditionally, the syphilis screening algorithm begins with a nontreponemal immunoassay, which is manually performed by a laboratory technologist. In contrast, the reverse algorithm begins with a treponemal immunoassay, which can be automated. The Centers for Disease Control and Prevention has recognized both approaches, but little is known about the current state of laboratory practice, which could impact test utilization and interpretation. Objective. To assess the current state of laboratory practice for syphilis serologic screening. Design. In August 2015, a voluntary questionnaire was sent to the 2360 laboratories that subscribe to the College of American Pathologists syphilis serology proficiency survey. Results. Of the laboratories surveyed, 98% (2316 of 2360) returned the questionnaire, and about 83% (1911 of 2316) responded to at least some questions. Twenty-eight percent (378 of 1364) reported revision of their syphilis screening algorithm within the past 2 years, and 9% (170 of 1905) of laboratories anticipated changing their screening algorithm in the coming year. Sixty-three percent (1205 of 1911) reported using the traditional algorithm, 16% (304 of 1911) reported using the reverse algorithm, and 2.5% (47 of 1911) reported using both algorithms, whereas 9% (169 of 1911) reported not performing a reflex confirmation test. Of those performing the reverse algorithm, 74% (282 of 380) implemented a new testing platform when introducing the new algorithm. Conclusion. The majority of laboratories still perform the traditional algorithm, but a significant minority have implemented the reverse-screening algorithm. Although the nontreponemal immunologic response typically wanes after cure and becomes undetectable, treponemal immunoassays typically remain positive for life, and it is important for laboratorians and clinicians to consider these assay differences when implementing, using, and interpreting serologic syphilis screening algorithms. (Arch Pathol Lab Med. 2017;141:93 97; doi: / CP) Accepted for publication June 2, Published as an Early Online Release October 20, From the Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio (Drs Rhoads and Ansari); the Department of Pathology, University of Utah, Salt Lake City (Dr Genzen); the Department of Surveys, College of American Pathologists, Northfield, Illinois (Ms Bashleben); and the Department of Pathology, Montefiore Medical Center, Bronx, New York (Dr Faix). The authors have no relevant financial interest in the products or companies described in this article. Reprints: M. Qasim Ansari, MD, Department of Laboratory Medicine, Cleveland Clinic, 9500 Euclid Ave, LL3, Cleveland, OH ( ansarim@ccf.org). Currently, no gold standard for immunologic syphilis screening and diagnosis is universally accepted, 1 and numerous assays are available for clinical testing. 2,3 Traditionally, the immunologic syphilis screening algorithm begins with a nontreponemal immunoassay (eg, rapid plasma reagin [RPR]), and if this result is positive, then the specimen is reflexed to an immunoassay for Treponemaspecific (treponemal) antibodies, which can serve as confirmation for the positive screen (Figure, A). 4,5 Historically, these immunoassays were manually performed and subjectively interpreted. Recently, advancements in instrumentation have enabled more automated and objective approaches to treponemal immunoassays. 6,7 With these advances, some laboratories have implemented a reverse syphilis screening algorithm (Figure, B), which uses an automated treponemal immunoassay as the initial syphilis screening test and subsequent confirmation with another immunoassay. 1,8 10 The advantages and disadvantages of the traditional and reverse algorithms have been discussed in other publications and will not be extensively discussed in this article. 11,12 In brief, the reverse algorithm is typically more expensive but is more amenable to automation, which can be advantageous in high-volume testing. Initial reports suggested the reverse algorithm may have lower specificity. 13,14 However, current literature has demonstrated that the reverse algorithm can be both highly specific and sensitive, with sensitivities Arch Pathol Lab Med Vol 141, January 2017 Traditional and Reverse Syphilis Screening Practices Rhoads et al 93
2 Traditionally, serologic syphilis screening has been performed using a nontreponemal screening assay and a treponemal confirmation test (A). More recently, some laboratories have implemented a reverse algorithm, which uses a treponemal test as the initial screening assay (B). Abbreviations: CIA, chemiluminescence immunoassay; EIA, enzyme immunoassay; FTA-Abs, fluorescent treponemal antibody absorption test; RPR, rapid plasma reagin assay; TP-PA, Treponema pallidum particle agglutination assay. surpassing that of the traditional algorithm The reverse algorithm is effective in detecting an initial syphilis infection, but because treponemal antibodies typically remain positive for life, immunologic assessment of reinfection is challenging. Diagnosing syphilis reinfection should include attention to the patient s history and a nontreponemal assay. Similarly, laboratory monitoring of syphilis treatment and recovery can only be accomplished by using a nontreponemal assay. Until now, few data have been published that describe how frequently these different algorithms are practiced in the clinical immunology laboratory. In this report we describe the syphilis screening practices of laboratories that subscribe to the College of American Pathologists (CAP) proficiency testing material for syphilis serology (Survey G). These data reveal the current laboratory practice and provide insight into how quickly syphilis testing is changing. The results demonstrate the high variability in test selection among laboratories and also reveal opportunities for some laboratories to improve their test recommendations in order to foster optimal test utilization. The Diagnostic Immunology Resource Committee is a volunteer group within the CAP that provides scientific and educational expertise in immunology for the CAP and its members. One of the committee s services to the CAP is to oversee the proficiency testing of Survey G. Recognizing the evolution in syphilis serology screening practices, the committee devised a comprehensive questionnaire for the Survey G participants in an attempt to determine the current state of syphilis serology screening in laboratory practice. MATERIALS AND METHODS In August of 2015, a voluntary supplemental 10-item questionnaire was included in the 2015 B mailing of the CAP Survey G, which was sent to 2360 laboratories in more than 50 countries. Some questions included the opportunity to choose other and then write in a response if the multiple choices were deemed insufficient by the participant. Answers of other were maintained as such and were not reclassified during the analysis. RESULTS Of the 2360 laboratories surveyed, 98.1% (2316) returned the questionnaire. Those that returned the questionnaire included 79.3% (1837 of 2316) from the United States, 1.9% (44 of 2316) from Canada, and 18.8% (435 of 2316) from other international laboratories. Of those that returned the questionnaire, 82.5% (1911 of 2316) responded to at least some of the questions (Table). Of those who responded, 81.1% (1550 of 1911) reported offering a single algorithm for syphilis serology screening, and 9.5% (181 of 1911) offered multiple algorithms depending on the patient demographics or clinician 94 Arch Pathol Lab Med Vol 141, January 2017 Traditional and Reverse Syphilis Screening Practices Rhoads et al
3 Questionnaire and Summary of Participant Responses a Question No. % 1. Does your laboratory offer a single algorithm for syphilis serology screening? Offer 1 screening algorithm for adults Offer multiple algorithms for adults depending on patient demographics and/or the preference of the ordering clinician Do not know Total Does your laboratory use a traditional algorithm (nontreponemal screen followed by treponemal confirmation) or a reverse algorithm (treponemal screen followed by nontreponemal confirmation)? Traditional algorithm Reverse algorithm Both traditional and reverse algorithm Lab does not reflexively perform a confirmation test Do not know Other Total If your laboratory uses the reverse algorithm, did your laboratory implement a new testing platform when initially implementing the reverse algorithm? Yes No Total Do not know b 126 n/a Our lab does not perform the reverse algorithm b 1254 n/a 4. Which testing method does your laboratory recommend using for its initial syphilis serology screening test? (Fill only one.) RPR, qualitative RPR with titer CMIA EIA total antibody Multiplex flow immunoassay EIA-IgG MHA-TP/TP-PA/PK-TP Other c Total We do not provide a specific recommendation b 54 n/a 5. If the test identified in question 4 is positive, which confirmation test is recommended and/or performed? (Fill only one.) FTA-ABS MHA-TP/TP-PA/PK-TP RPR with titer EIA-IgG RPR, qualitative Our lab recommends using 2 or more confirmation tests Other d Total Not applicable b 121 n/a 6. Is the confirmation test described in question 5 performed in your laboratory (ie, same CLIA number) or sent out to another laboratory? Performed in our lab Sent to another lab Other Total Not applicable b 96 n/a 7. Does your laboratory recommend using a specific test to monitor the success of syphilis treatment? Yes No Continued Question No. % Do not know Total If you answered yes to question 7, which test does your laboratory recommend? RPR with titer VDRL with titer FTA-ABS MHA-TP/TP-PA/PK-TP RPR, qualitative Other e Our lab recommends monitoring with more than one test Total Not applicable, lab does not recommend using a specific test to monitor the success of syphilis treatment b 746 n/a 9. When was the last time your laboratory s syphilis serology testing algorithm was revised? Less than 1 year ago Between 1 and 2 years ago More than 2 years ago Total Do not know b 534 n/a 10. Does your laboratory anticipate changing its serology testing algorithm in the next 12 months? Yes No Do not know Total Abbreviations: CLIA, Clinical Laboratory Improvement Amendments; CMIA, chemiluminescent microplate immunoassay; EIA, enzyme immunoassay; FTA-ABS, fluorescent treponemal antibody absorption test; TP-LIA, Treponema pallidum line immunoassay; MHA-TP, microhemagglutination assay for Treponema pallidum antibodies; PK-TP, Olympus automated hemagglutination test; RPR, rapid plasma reagin assay; TP-PA, Treponema pallidum particle agglutination assay; VDRL, venereal disease research laboratory test; USR, unheated serum reagin test. a Most responses comprising less than 2.0% of the total were combined with other in this table. b Results not included in total number of responses for question-specific percentages. c Responses in this group include the following: VDRL with titer, qualitative VDRL, FTA-ABS, USR with titer, TP-LIA, and other. d Responses in this group include the following: VDRL with titer, EIA total antibody, CMIA, multiplex flow immunoassay, TP-LIA, qualitative VDRL, qualitative USR, USR with titer, and other. e Responses in this group include the following: CMIA, USR with titer, TP-LIA, EIA total antibody, multiplex flow immunoassay, EIA-IgG, and other. preference (question 1). A total of 9.4% (180 of 1911) of respondents reported they did not know if a single algorithm was offered by their laboratory. When asked if their laboratory uses the traditional or reverse algorithm (question 2), 63.1% (1205 of 1911) reported using the traditional algorithm, 15.9% (304 of 1911) reported using the reverse algorithm, and 2.5% (47 of 1911) reported using both algorithms. A total of 8.8% (169 of 1911) of respondents noted that their laboratory does not reflexively perform a confirmation test. The remainder of respondents either did not know or chose other. Other write-in responses specified that some laboratories (1) send confirmation testing to a reference laboratory, (2) perform confirmation testing according to each clinician s preference, (3) perform different algorithms for different patient groups (eg, prenatal patients, inpatients, organ donors), (4) choose a confirmatory test to complement the initial test Arch Pathol Lab Med Vol 141, January 2017 Traditional and Reverse Syphilis Screening Practices Rhoads et al 95
4 ordered (eg, nontreponemal assay is confirmed with treponemal assay and vice versa) or (5) use a modified algorithm (eg, multiple assays used to confirm a positive screen) (qualitative data not shown). Of the laboratories that perform the reverse algorithm (question 3), 74% (282 of 380) reported implementing a new testing platform when introducing the reverse algorithm, and 26% (98 of 380) did not introduce a new platform. Of the laboratories that recommend a specific assay for initial syphilis serology screening (question 4), about threequarters recommend an RPR assay. A total of 41.7% (783 of 1876) recommend a qualitative RPR and 32.4% (607 of 1876) recommend an RPR with titer. Other initial screening assays that are commonly recommended by laboratories include a chemiluminescent microplate immunoassay (181 of 1876; 9.6%), an enzyme immunoassay for total antibodies (90 of 1876; 4.8%) or only IgG (55 of 1876; 2.9%), a multiplex flow assay (57 of 1876; 3.0%), an agglutination (Treponema pallidum particle agglutination assay) or hemagglutination assay (microhemagglutination assay for T pallidum antibodies or Olympus automated hemagglutination test) (39 of 1876; 2.1%), or the venereal disease research laboratory test (36 of 1876; 1.9%). Confirmation testing that the laboratories recommended and/or performed reflexively after a positive initial screen (question 5) includes 34.7% (615 of 1774) that use the fluorescent treponemal antibody-absorption test, 23.1% (410 of 1774) that use an agglutination or hemagglutination assay, and 23.2% (412 of 1774) that use an RPR assay. More rarely used confirmation tests include enzyme immunoassay for total antibodies (27 of 1774; 1.5%) or only IgG (48 of 1774; 2.7%), venereal disease research laboratory test with titer (34 of 1774; 1.9%), and chemiluminescent microplate immunoassay (21 of 1774; 1.2%). A total of 5.0% (89 of 1774) of laboratories reported using 2 or more confirmation tests, and 4.6% (82 of 1774) of laboratories chose other, with many of the open-ended other responses describing sending the specimen to a public health or reference laboratory for confirmation testing. Of those laboratories for which confirmatory testing is recommended and/or performed, only 36.6% (663 of 1813) of laboratories perform a confirmation test in their own laboratory (question 6), whereas 61.8% (1121 of 1813) of the laboratories send confirmation testing to a different laboratory. When the participants were asked if their laboratory recommends the use of a specific assay to monitor the success of syphilis treatment (question 7), 23.5% (448 of 1907) responded yes and 58.4% (1113 of 1907) responded no. Some of the respondents (346 of 1907; 18.1%) did not know if their laboratory had a specific recommendation. Of those laboratories that do recommend an assay for monitoring treatment (question 8), RPR with titer is recommended by the majority of respondents (330 of 462; 71.4%). Other recommendations include venereal disease research laboratory test with titer (38 of 462; 8.2%), fluorescent treponemal antibody-absorption test (35 of 462; 7.6%), an agglutination or hemagglutination assay (16 of 462; 3.5%), qualitative RPR (14 of 462; 3.0%), and rarely other assays. Seven laboratories (1.5%) recommend monitoring with more than one test. When asked when their laboratory s syphilis serology testing algorithm was last revised (question 9), 534 of 1898 respondents did not know. Of those that did know, 14.9% (203 of 1364) reported the algorithm had been revised within the last year, 12.8% (175 of 1364) reported revision between 1 and 2 years ago, and 72.3% (986 of 1364) reported revision more than 2 years ago. When the participants were asked if they anticipate their laboratory changing its algorithm in the next 12 months (question 10), 8.9% (170 of 1905) replied yes, 63.7% (1213 of 1905) replied no, and 27.4% (552 of 1905) did not know. DISCUSSION This report describes the current state of laboratory practice for syphilis screening among laboratories that subscribe to the CAP proficiency testing Survey G. Approximately 80% (1550 of 1911) of the laboratories that responded to our poll offer a single algorithm, and about 80% (1205 of 1550) of these continue to offer the traditional screening algorithm. About 20% (304 of 1550) use the newer reverse algorithm in their laboratory practice. Although the questionnaire did not inquire as to the volume of specimens analyzed in each laboratory, we hypothesize that largervolume laboratories may be more quickly adopting the reverse algorithm than lower-volume laboratories because higher-volume laboratories may more substantially improve workflow through automating syphilis screening. It is appropriate for laboratories that offer syphilis serology screening to recommend an algorithm to ordering clinicians in order to facilitate proper test utilization, and it is important that confirmatory testing be performed in a timely manner. However, many laboratories refer specimens to a reference laboratory for confirmatory testing, which inherently incurs some delay between reporting a positive screening result and reporting confirmation testing results. It may not be surprising that many laboratories still use the traditional approach and still use the RPR assay as the initial screening test. There is still controversy regarding the newer reverse algorithm, especially with regard to specimens that are negative when confirmatory nontreponemal testing is performed. Some experts argue that these represent false-positive screens because antitreponemal antibodies may persist long after treatment has eradicated the patient s infection. Others consider the nontreponemal testing to be a false-negative confirmatory result, especially if tertiary disease (or neurologic involvement) is present. When treponemal and nontreponemal assay results disagree, performance of a second treponemal assay may help to resolve the discrepancy. There may be a slow movement toward adoption of the newer approach. At the time of the survey, about onequarter of laboratories had revised their syphilis screening algorithm within the past 2 years, and about 10% (170 of 1905) were anticipating changes in the coming year. This likely represents a move toward the reverse algorithm. Also, the 2014 update of the syphilis management guideline from the International Union against Sexually Transmitted Infections in Europe recommends using a reverse algorithm because of its increased sensitivity. 18 The European guideline also describes the potential use of algorithm iterations that have not been discussed in this report, such as confirming a positive treponemal screening assay with a second treponemal assay or performing both a nontreponemal and a treponemal assay on all samples sent for screening. It should be noted that the Centers for Disease Control and Prevention describes the possibility of using either the traditional or the reverse algorithm, but the Centers for Disease Control and Prevention recommends that no matter which algorithm is used, both treponemal 96 Arch Pathol Lab Med Vol 141, January 2017 Traditional and Reverse Syphilis Screening Practices Rhoads et al
5 and nontreponemal assays be included in the diagnostic testing algorithm for syphilis. 19 Antitreponemal antibodies typically persist for life, so they are not suitable for monitoring disease. Those performing syphilis testing should recognize that RPR titer is the cornerstone of monitoring disease after syphilis diagnosis, and RPR is the primary assay used for diagnosing reinfection. We recommend that these best practices be used by all laboratories and that all laboratories work to ensure both testing personnel and individuals responding to clinician queries recognize these principles of syphilis serology. Numerous serologic assays are available for syphilis testing, and multiple screening algorithms have been proposed. It is important for laboratorians to be familiar with emerging practices in syphilis screening, so they can make informed decisions regarding test implementation, utilization, and interpretation. As always, effective communication between the laboratory health care providers and primary health care providers is essential in providing effective and appropriate patient testing and care. References 1. Morshed MG, Singh AE. Recent trends in the serologic diagnosis of syphilis. Clin Vaccine Immunol. 2015;22(2): Cole MJ, Perry KR, Parry JV. Comparative evaluation of 15 serological assays for the detection of syphilis infection. Eur J Clin Microbiol Infect Dis. 2007; 26(10): Binnicker MJ, Jespersen DJ, Rollins LO. Treponema-specific tests for serodiagnosis of syphilis: comparative evaluation of seven assays. J Clin Microbiol. 2011;49(4): Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev. 1995;8(1): Centers for Disease Control and Prevention (CDC). Syphilis testing algorithms using treponemal tests for initial screening four laboratories, New York City, MMWR Morb Mortal Wkly Rep. 2008:57(32); Accessed February 22, Marangoni A, Moroni A, Accardo S, Cevenini R. Laboratory diagnosis of syphilis with automated immunoassays. J Clin Lab Anal. 2009;23(1): Marangoni A, Nardini P, Foschi C, et al. Evaluation of the BioPlex 2200 syphilis system as a first-line method of reverse-sequence screening for syphilis diagnosis. Clin Vaccine Immunol. 2013;20(7): Binnicker MJ, Jespersen DJ, Rollins LO. Direct comparison of the traditional and reverse syphilis screening algorithms in a population with a low prevalence of syphilis. J Clin Microbiol. 2012;50(1): Sena AC, White BL, Sparling PF. Novel Treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century. Clin Infect Dis. 2010; 51(6): Loeffelholz MJ, Binnicker MJ. It is time to use treponema-specific antibody screening tests for diagnosis of syphilis. J Clin Microbiol. 2012;50(1): Owusu-Edusei K, Peterman TA, Ballard RC. Serologic testing for syphilis in the United States: a cost-effectiveness analysis of two screening algorithms. Sex Transm Dis. 2011;38(1): Binnicker MJ. Which algorithm should be used to screen for syphilis? Curr Opin Infect Dis. 2012;25(1): Centers for Disease Control and Prevention (CDC). Discordant results from reverse sequence syphilis screening five laboratories, United States, MMWR Morb Mortal Wkly Rep. 2011;60(5); mmwr/pdf/wk/mm6005.pdf. Accessed February 22, Lipinsky D, Schreiber L, Kopel V, Shainberg B. Validation of reverse sequence screening for syphilis. J Clin Microbiol. 2012;50(4): Tong ML, Lin LR, Liu LL, et al. Analysis of 3 algorithms for syphilis serodiagnosis and implications for clinical management. Clin Infect Dis. 2014; 58(8): Huh HJ, Chung JW, Park SY, Chae SL. Comparison of automated treponemal and nontreponemal test algorithms as first-line syphilis screening assays. Ann Lab Med. 2016;36(1): Park BG, Yoon JG, Rim JH, Lee A, Kim HS. Comparison of six automated treponema-specific antibody assays. J Clin Microbiol. 2016;54(1): Janier M, Hegyi V, Dupin N, et al European guideline on the management of syphilis. J Eur Acad Dermatol Venereol. 2014;28(12): Workowski KA, Bolan GA. Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep. 2015;64(RR-03): cdc.gov/std/tg2015/tg-2015-print.pdf. Accessed February 22, Arch Pathol Lab Med Vol 141, January 2017 Traditional and Reverse Syphilis Screening Practices Rhoads et al 97
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