Guidelines for Serological Testing for Syphilis Sex Transm Infect: First Published As 10.1136/Sti.76.5.403 on 1 October 2000

Sex Transm Inf 2000;76:403–405 403

Guidelines for serological testing for syphilis Sex Transm Infect: first published as 10.1136/sti.76.5.403 on 1 October 2000. Downloaded from

Diagnostics H Young

Although we may wish it were not so, syphilis, an EIA (quoted with permission of the UK like the poor, will always be with us—at least NEQAS organiser). Advantages of EIA include for the foreseeable future. The levels of both automated (or semiautomated) processing, are determined to a large extent, by political objective reading of results, and interfacing instability and socioeconomic deprivation. with the laboratory computer system to allow Overall, the incidence of syphilis is low in electronic report generation. The widespread Western Europe (approximately 0.3 cases/ and growing use of automation and computeri- 100 000 in England in 1998)1 although it has sation in laboratories has led to the reorganisa- reached epidemic proportions in the Russian tion and rationalisation of microbial serology to Federation where the levels in 1996 exceeded meet the continuous demands for increased 900 cases/100 000 in men and women in the cost eVectiveness in service delivery. Several 20–29 year old age group.2 The need to main- requests to the author for advice suggest that, tain eVective strategies for syphilis control, owing to rationalisation of services and devel- which must include diagnosis and manage- opments in automation and computerisation, ment, in areas of low prevalence such as the there is a trend for syphilis testing to move from United Kingdom, is reinforced by the recent bacteriology laboratories to dedicated micro- local outbreak of heterosexually acquired bial serology laboratories that traditionally may syphilis in South West England3 as well as the have dealt mainly with viral serology. These marked increase in homosexually acquired changes make the guidelines particularly timely infection in the Manchester area.4 A significant as many laboratories may be taking on syphilis proportion of the infected men in Manchester testing for the first time. There is also a trend were HIV positive so the overall community for fewer laboratories to perform confirmatory health gain from rapid and eVective diagnosis testing, preferring to forward specimens reac- extends well beyond syphilis: ulcerative sexu- tive on screening to a specialised laboratory or ally transmitted infections promote HIV trans- centre. mission by augmenting HIV infectiousness and The guidelines outline the current serologi- HIV susceptibility via a variety of biological cal tests for syphilis and highlight the diVer- mechanisms.5 The importance of the serologi- ences in screening practice between the United cal diagnosis of syphilis has now been recog- Kingdom and the United States. They contain nised with the publication of the excellent an excellent algorithm for “Treponemal anti- guidelines for serological testing for syphilis in body screening and confirmatory testing” http://sti.bmj.com/ diagnostic microbiology laboratories by the which is based on the key recommendations of PHLS Syphilis Serology Working Group.6 the group. The guidelines recognise that there These complement the recent national guide- are a number of commercial tests of any given lines on the management of syphilis78 and format and that these can vary in performance together they should improve the overall diag- characteristics. Decisions on which test a labo- nosis and management of syphilis within the ratory uses will be based on many factors

United Kingdom and beyond. including cost, ease of use, suitability for auto- on September 27, 2021 by guest. Protected copyright. Guidelines for serological diagnosis for mation, compatibility with the format of other syphilis are long overdue. The last guidelines tests already in use in the laboratory, as well as which were produced by the World Health performance characteristics. Sadly, the changes Organization in 19829 recommended the use of described above inevitably decrease the weight- a cardiolipin antigen test such as the Venereal ing of performance characteristics in test selec- Diseases Reference Laboratory (VDRL) or tion. There is an enormous choice of test rapid plasma reagin (RPR) test and the reagents, manufactured and/or supplied by dif- Treponema pallidum haemagglutination assay ferent companies. For example, of the UK (TPHA) for screening for syphilis. The new laboratories participating in the NEQAS recommendations6 extend the WHO guide- scheme there were 19 diVerent cardiolipin lines by suggesting that treponemal antigen tests, 13 TPHA/TPPA tests, nine EIAs, and based enzyme immunoassays (EIAs) are an seven FTA-abs in use (quoted with permission Department of appropriate alternative to the combined of the UK NEQAS organiser). It is important Medical Microbiology, VDRL/RPR and TPHA screen. EIA as a single that laboratories do not change reagents Edinburgh University screening test was shown to give similar results frequently in order that they and their users Medical School, Teviot to the VDRL/RPR and TPHA combination such as genitourinary medicine physicians Place, Edinburgh 10 EH8 9AG, UK some years ago and is already used by many become fully conversant with the performance H Young laboratories, particularly those with large characteristics of the particular tests used. workloads. Results of the UK National Exter- Published performance criteria following strin- Correspondence to: nal Quality Assessment Scheme for Microbiol- gent evaluation in independent centres are [email protected] ogy syphilis serology distribution in April 2000 available for very few of the numerous tests Accepted for publication showed that 56% (130/232) of responding (and their modiﬁcations) produced by diVerent 8 September 2000 laboratories within the United Kingdom used manufacturers. For example, there is a paucity

www.sextransinf.com 404 Young

of performance data for the various TPHA kits, adequate treatment, a negative VDRL result, Sex Transm Infect: first published as 10.1136/sti.76.5.403 on 1 October 2000. Downloaded from supplied by 12 diVerent companies, used in the like a negative IgM, does not exclude the need United Kingdom. There are also few evalua- for treatment: in one study16 all 33 patients with tions of the Treponema pallidum particle agglu- untreated late latent syphilis gave a negative tination assay (TPPA), which uses gelatin par- Captia Syphilis-M EIA result. Group (c) is the ticles as a carrier rather than erythrocytes, and only group where additional confirmatory test- has replaced the TPHA in many laboratories. ing is recommended. Where the first line con- An early developmental report11 found that the firmatory treponemal antigen test is negative microcapsule agglutination method was supe- and the additional confirmatory treponemal rior to the THPA in detecting cases of primary antigen test(s) and VDRL are negative then the syphilis. More recently, Pope and colleagues in specimen can be reported as a false positive the United States12 reported that the TPPA was screening test. If at least one additional an appropriate substitute for the TPHA. confirmatory test is reactive then this signifies a Another recent report found that the TPPA low level of treponemal antibodies, which was significantly more sensitive than the FTA- could be the result of a treated or longstanding abs and marginally more sensitive than the infection or to an early primary infection. IgM TPHA,13 which makes the TPPA very suitable EIA testing will diVerentiate between these as a confirmatory test. possibilities. If the only reactive screening test The starting point for the algorithm is the were the VDRL/RPR, and primary syphilis was result of screening (either VDRL/RPR and not suspected, the negative result in the TPHA or EIA alone). Screening with a treponemal confirmatory test normally used non-treponemal test alone is not recom- would seem suYcient to denote a biological mended because of the potential for false nega- false positive reaction (if quantitative VDRL/ tive results as a result of the prozone RPR positive) or a false positive VDRL/RPR phenomenon.14 A negative screening result is screening test (if quantitative VDRL/RPR reported as “Treponemal antibody NOT de- negative). Again, an IgM EIA test should be tected but advise repeat if at risk of recent performed when primary syphilis is suspected. infection.” The importance of repeat testing is In all categories of treponemal infection a well founded because approximately 15% of repeat specimen is advised to confirm the find- patients with primary syphilis will be seroneg- ings; this will normally be a week or so after the ative at initial presentation.15 A reactive screen- initial specimen. It is also recommended that ing result should be confirmed with a trepone- all reactive specimens should be referred to one mal antigen test diVerent from that used in of the PHLS syphilis specialist laboratories screening (for example, TPHA if EIA is used listed at the end of the guidelines for reference for screening) and a quantitative non- testing and to allow collection of surveillance treponemal test (VDRL/RPR). The various data. scenarios which are then dealt with in detail While the detection of early infectious syphi- include: (a) confirmatory and non-treponemal lis is the priority of any syphilis testing tests reactive; (b) confirmatory test reactive but programme it must also be recognised that in non-treponemal test negative; and (c) con- the United Kingdom most newly diagnosed http://sti.bmj.com/ firmatory test negative with a negative or reac- cases are late stage infection.1 The screening tive non-treponemal test. Group (a) is the schedules proposed in the guidelines take group most likely to include untreated syphilis account of this and achieve high sensitivity in (or other treponemal disease) at any stage and all stages of infection. However, depending on it is suggested that an EIA for specific the particular tests used and the quality of the antitreponemal IgM should be considered on clinical/laboratory liaison there may be a failure

the basis of non-treponemal test titre and clini- to detect a small proportion of untreated on September 27, 2021 by guest. Protected copyright. cal details. Positive IgM reactions are consid- primary infections at one end of the spectrum ered to be consistent with recent/active and markers of long standing treated trepone- treponemal infection: the commercially avail- mal infection at the other. The extent of these able Captia Syphilis-M EIA has a sensitivity of failures will vary slightly and depend on the 93% in primary infection, 85% in secondary particular tests used. By the time that signs and infection, and 64% in early latent infection.16 symptoms of primary syphilis are present most However, it is noted that in the absence of a patients have detectable IgG and IgM18 but history of adequate treatment, a negative result before this there is a short window around 2–4 does not exclude the need for treatment. weeks post-infection when only IgM is detect- Irrespective of the IgM result VDRL titres able. Provided that the specificity of EIAs that greater than 16 are rarely found in adequately detect both IgM and IgG is as good as those treated infections.17 Sera in group (b) are most that detect only IgG, and the sensitivity in late likely to be from treated patients or those with stage infection is also as good, it would be an untreated late latent infection. Occasionally, advantage to use the former type of EIA even however, sera from patients with primary although primary syphilis is rare. Specificity is infection may give this pattern: I am aware of important in terms of the cost and workload two recent cases of primary syphilis where the involved in confirmatory testing and referral: a VDRL test was negative yet the screening EIA 0.5% decrease in specificity of a test used in a was positive. Although the algorithm does not laboratory screening 20 000 specimens per include IgM testing of this group, IgM testing year means an extra 100 referrals for confirma- should be undertaken in all cases of suspected tion at a cost of around £1500 to £3000. primary infection irrespective of the initial Even when highly specific screening tests are screening results. In the absence of a history of used confirmatory testing is essential because

www.sextransinf.com Guidelines for serological testing for syphilis 405

of the low prevalence of syphilis. For example, centres. Such changes would also have enor- Sex Transm Infect: first published as 10.1136/sti.76.5.403 on 1 October 2000. Downloaded from treponemal EIAs generally give a specificity on mous benefit for the prompt surveillance of screening of around 99.5%. However, if the infectious syphilis. Further guidelines dealing prevalence of syphilis in the test population is with specific areas of syphilis serology such as 0.5% (it will be lower in populations such as congenital infection, neurosyphilis and co- antenatal patients) then the predictive value of existing HIV are promised.6 Guidance on a positive screening test result is only 50%. direct detection of Treponema pallidum in geni- Applying a confirmatory test with the same tal ulcers would also be welcome. specificity to the sera reactive on screening (prevalence of syphilis now 50% in this 1 Lamagni TL, Hughes G, Rogers PA, et al. New cases seen at population) will give a positive predictive value genitourinary medicine clinics: England 1998. Commun Dis of 99%. The guideline recommendation that Rep Suppl 6 1999;9:S1–12 2 Tichonova L, Borisenko K, Ward H, et al. Epidemics of specimens that are reactive on screening syphilis in the Russian Federation: trends, origins, and pri- require confirmatory testing with a diVerent orities for control. Lancet 1997;350:210–13. 3 Battu VR, Horner PJ, Taylor PK, et al. Locally acquired het- treponemal test, of equal sensitivity, from that erosexual outbreak of syphilis in Bristol. Lancet 1997;350: used for screening and, ideally, greater specifi- 1100–1. 4 CDSC. Increased transmission of syphilis in Manchester. city highlights the shortcomings of the FTA- Commun Dis Rep CDR Wkly 2000;10:89. abs, previously considered the “gold standard” 5 Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: the contribution of confirmatory test. Indeed the FTA-abs is not other sexually transmitted diseases to sexual transmission recommended as the first line confirmatory of HIV infection. Sex Transm Inf 1999;75:3–17. 6 Egglestone SI, Turner AJL, for the PHLS Syphilis Serology test. The specificity of the FTA-abs is poorer Working Group. Serological diagnosis of syphilis. Commun than that of the other treponemal antigen Dis Public Health 2000;3:158–62. 17 19 7 Clinical EVectiveness Group (Association of Genitourinary screening tests while certain newer EIAs are Medicine and the Medical Society for the Study of significantly more sensitive than the Venereal Diseases). National guidelines for the manage- 13 20 ment of early syphilis. Sex Transm Inf 1999;75:S29–33. FTA-abs in detecting markers of past infec- 8 Clinical EVectiveness Group (Association of Genitourinary tion which means that the FTA-abs will fail to Medicine and the Medical Society for the Study of Venereal Diseases). National guidelines for the manage- confirm a small number of genuinely reactive ment of late syphilis. Sex Transm Inf 1999;75:S34–7 EIAs. The TPHA/TPPA is comparable with 9 World Health Organization. Treponemal infections. Technical report series 674. Geneva: WHO, 1982. the newer EIAs which means that the most 10 Young H, Moyes A, McMillan A, et al. Screening for accurate confirmation of treponemal antibod- treponemal infection by a new enzyme immunoassay. Geni- tourin Med 1989;65:72–8. ies will result from using the TPHA/TPPA to 11 Kobayashi S, Yamaya SI, Sugahara T, et al. Microcapsule confirm a reactive EIA or an EIA to confirm a agglutination test for T pallidum antibodies. A new serodi- agnostic test for syphilis. Br J Vener Dis 1983;59:1–7. reactive TPHA/TPPA: the practicalities of 12 Pope V, Fears MB, Morrill WE, et al. Comparison of the laboratory testing mean that the former Serodia Treponema pallidum particle agglutination, Captia-Syphilis-G, and SpiroTek Reagin II tests with scenario is more likely. Although the FTA-abs standard test techniques for diagnosis of syphilis. J Clin may have slightly greater sensitivity in early Microbiol 2000;38:2543–5. 13 Young H, Aktas G, Moyes A. Enzywell recombinant enzyme primary infection a positive FTA-abs result immunoassay for the serological diagnosis of syphilis. Int J alone has poor specificity and in one study STD AIDS 2000;11:288–91. 14 Jurado RL, Campbell J, Martin PD. Prozone phenomenon more than 90% of such reactions were the in secondary syphilis Has its time arrived? Arch Intern Med result of conditions other than syphilis.21 The 1993;153:2496–8. http://sti.bmj.com/ 15 Anderson J, Mindel A, Tovey SJ, et al. Primary and second- use of an anti-treponemal IgM to supplement ary syphilis, 20 years’ experience. 3: Diagnosis, treatment, standard screening and confirmatory proce- and follow up. Genitourin Med 1989;65:239–43. 16 Lefevre JC, Bertrand MA, Bauriaud R. Evaluation of the dures is a better approach to maximising the Captia enzyme immunoassays for detection of immu- detection of early primary infection than noglobulins G and M to Treponema pallidum in syphilis. J Clin Microbiol 1990;28:1704–7. relying on the FTA-abs. Other limitations of 17 Luger AFH. Serological diagnosis of syphilis: current meth- the FTA-abs in initial confirmation include the ods. In: Young H, McMillan A, eds. Immunological diagnosis of sexually transmitted diseases. New York and Basle: Marcel finding that false reactivity in the FTA-abs was Dekker, 1988:249–74. on September 27, 2021 by guest. Protected copyright. significantly associated with false reactive EIA 18 Baker-Zander SA, Hook EW, Bonin P, et al. Antigens of 22 Treponema pallidum recognized by IgG and IgM antibod- results ; and the subjective interpretation of ies during syphilis in humans. J Infect Dis 1985;151:264– the test that may lead to bias when other test 72. 23 19 Young H. Syphilis: new diagnostic directions. Int J STD results are known. AIDS 1992;3:391–413. These guidelines will not only be of immedi- 20 Young H, Moyes A, Seagar L, et al. Novel recombinant- antigen enzyme immunoassay for serological diagnosis of ate benefit to laboratories involved in syphilis syphilis. J Clin Microbiol 1998;36:913–7. serology they will also act as an impetus to 21 Carlsson B, Hanson HS, Wasserman J, et al. Evaluation of the fluorescent treponemal antibody-absorption (FTA- review the provision of syphilis serology Abs) test specificity. Acta DermVenereol (Stockh) 1991;71: services throughout the United Kingdom. 306–11. 22 Chronas G, Moyes A, Young H. Syphilis diagnosis: screen- Review could lead to improvements in reliabil- ing by enzyme immunoassay and variation in fluorescent ity and cost eVectiveness of laboratory testing antibody absorbed (FTA-abs) confirmatory test performance. Med Lab Sci 1992;49:50–5. via increased standardisation of screening tests 23 Halling VW, Jones MF, Bestrom JE, et al. Clinical compari- with clearly identified policies for referral of son of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal confirmatory tests to appropriately resourced antibody absorption immunoglobulin G assay for syphilis regional or supraregional specialist/reference testing. J Clin Microbiol 1999;37:3233–4.