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Evaluation of the sensitivity and specificity of a Treponema pallidum dried blood spot technique for use in the detection of syphilis
TRANSACTIONS
OFTHE
ROYAL
SOCIETY
OFTROPICAL
MEDICINE
Evaluation of the sensitivity and specificity of a Treponema pallidurn dried blood spot technique for use in the detection of syphilis G. L. Coatesl, L. Guarentil, S. l? Parker?-, J. F. Willumsenl and A. M. Tomkins’ ‘Centre for International Child Health, Institute of Child Health, 30 Guilford Street, London, WClN lEH, UK; 2Department of Virology, Great Ormond Street Hospital for Sick Children NHS Trust, Great Ormond Street, London, WClN 3JH UK syphilis, Treponemapallidurn, diagnosis, particle agglutination, dried blood spots
Keywords:
Early treatment and prevention of congenital syphilis relies on access to antenatal screening for syphilis antibodies and effective programmes require an easy and economical method of testing that is both sensitive and specific. The use of dried blood spots for antibody testing can meet these criteria as they are easier and more economical to collect, store and transport than serum. An enzyme-linked immunosorbent assay for syphilis has been adapted previously for use with blood spots (tiEVENS et al., 1992); however, this method reouires a plate reader which renders the test unsuitable for-use in iaboratories with limited facilities. We have modified a commercially available specific Treponema pallidurn particle agglutination (TPPA 2 test ?.designed for serological testing (Serodia TPPA : Fulu-ebio, Japan), for use with blood spots. The test uses gelatin particles sensitized with T. pallidurn antigen rather than the sensitized avian erythrocytes used in conventional T. pallidum specific haemagglutination tests (TPHA). These particles minimize non-specific agglutination and are more robust in adverse conditions. Unlike tests which are non-specific for T. pallidurn, e.g. the rapid plasma reagin test &l?R), the TPPA test will not produce false positive results with other tissue-damaging diseases (LARSEN et al., 1995). In 1995, 1037 matched serum samples and blood spots were collected from women attending routine antenatal clinics in rural Tanzania. The whole blood was immediatelv suotted on to Guthrie cards (Her Maiestv’s Stationery bmce, London, UK) and allowed to air-dry for 24 h before storage in sealed bags at 4°C until tested. At the same time the corresponding serum sample was tested by the RPR test (Rando+, UK). Courses of penicillin were given to women with positive RPR results. The sera were then transported to London where they were stored at 40°C. A 55mm diameter disk was punched out from the blood on the Guthrie card and placed in the well of a
AND
HYGIENE
(1998) 92,44
flat-bottomed microtitre plate (Dynatech,USA); l?O.& of elution buffer @phosphate-buffered saline contammg 0.05% Tween 80 ,0.005% sodium azide, pH 7.2) were added to each well and the plates were shaken for 5 min and then left to elute overnight at 4°C. The plates were shaken for 2 min before testing the eluate. A 1:5 dilution of each eluate was made in TPHA buffer (Fuiirebio, Tapan) and 25 l.tL were transferred to a V-well’micro&e plate (Dvnatech, USA). The TPPA test particles were reconstituted according to the manufacturer’s instructions and subsequently diluted 1: 10 with TPHA buffer immediately before use; 25 & of diluted test particles were added to each well (including kit controls, which were not prediluted). The plates were shaken briefly for 10 s and incubated for 90 min at room temperature on a flat, vibration-free surface. Plates were visually evaluated on a lightbox: a negative result was shown by the formation of a tight discrete button, whilst positive results were indicated by clumps of particles with agglutinated edges. In order to assess the sensitivity and specificity of the TPPA, we tested sera with the Serodia Tl?PAa test, according to the manufacturer’s instructions, and compared blood spots and serological results. Among the 1037 samples, 59 were positive by TPPA when using sera and 58 were TPPA positive when using the blood spots. Thus the modified blood spot TPPA test had a sensitivity of 98.3%. None of the positive TPPA blood spot samples was found to be negative by serological testing, giving the modified blood spot TPPA technique a specifity of 100%. The very high sensitivity and specificity of the blood suet TPPA test. comnared to the use of serum. make it acceptable for use as a specific test for T. pallidurn antibodies. Our modification of the existing test for use with blood spots is simple and does not complicate the test procedure. Blood spots can be obtained by finger-prick and the samples can be stored at room temperature, thereby reducing collection, storage and transportation problems.The modified blood spot TPPA test is more economical than most non-treponemal tests (LO. 10 per sample compared with LO.40 per sample for an RPR test). It has the additional advantages of being more sensitive and specific than non-treponemal tests, reducing the risk of false results, and it is easy to perform. The test is therefore appropriate for use in low resource settings, with limited laboratory facilities, for either routine clinical screening or large epidemiological surveys for syphilis. I
I
I
Larsen, S. A., Steiner, B. M. & Rudolph, A. H. (1995). Laboratory diagnosis and interpretation of tests for syphilis.CZinical Microbiolopv Reviews,8, l-2 1. Stevens, R., Pa&, K., Fuller; S., Wiznia, A., Noble, L., Duva, S. &Neal, M. (1992). Blood spot screening and confirmatory tests for syphilis antibody. Journal of Clinical Microbiology, 30,2353-2358.
Received 3July 1997; acceptedforpublication
11 July 1997
OFTHE
ROYAL
SOCIETY
OFTROPICAL
MEDICINE
Evaluation of the sensitivity and specificity of a Treponema pallidurn dried blood spot technique for use in the detection of syphilis G. L. Coatesl, L. Guarentil, S. l? Parker?-, J. F. Willumsenl and A. M. Tomkins’ ‘Centre for International Child Health, Institute of Child Health, 30 Guilford Street, London, WClN lEH, UK; 2Department of Virology, Great Ormond Street Hospital for Sick Children NHS Trust, Great Ormond Street, London, WClN 3JH UK syphilis, Treponemapallidurn, diagnosis, particle agglutination, dried blood spots
Keywords:
Early treatment and prevention of congenital syphilis relies on access to antenatal screening for syphilis antibodies and effective programmes require an easy and economical method of testing that is both sensitive and specific. The use of dried blood spots for antibody testing can meet these criteria as they are easier and more economical to collect, store and transport than serum. An enzyme-linked immunosorbent assay for syphilis has been adapted previously for use with blood spots (tiEVENS et al., 1992); however, this method reouires a plate reader which renders the test unsuitable for-use in iaboratories with limited facilities. We have modified a commercially available specific Treponema pallidurn particle agglutination (TPPA 2 test ?.designed for serological testing (Serodia TPPA : Fulu-ebio, Japan), for use with blood spots. The test uses gelatin particles sensitized with T. pallidurn antigen rather than the sensitized avian erythrocytes used in conventional T. pallidum specific haemagglutination tests (TPHA). These particles minimize non-specific agglutination and are more robust in adverse conditions. Unlike tests which are non-specific for T. pallidurn, e.g. the rapid plasma reagin test &l?R), the TPPA test will not produce false positive results with other tissue-damaging diseases (LARSEN et al., 1995). In 1995, 1037 matched serum samples and blood spots were collected from women attending routine antenatal clinics in rural Tanzania. The whole blood was immediatelv suotted on to Guthrie cards (Her Maiestv’s Stationery bmce, London, UK) and allowed to air-dry for 24 h before storage in sealed bags at 4°C until tested. At the same time the corresponding serum sample was tested by the RPR test (Rando+, UK). Courses of penicillin were given to women with positive RPR results. The sera were then transported to London where they were stored at 40°C. A 55mm diameter disk was punched out from the blood on the Guthrie card and placed in the well of a
AND
HYGIENE
(1998) 92,44
flat-bottomed microtitre plate (Dynatech,USA); l?O.& of elution buffer @phosphate-buffered saline contammg 0.05% Tween 80 ,0.005% sodium azide, pH 7.2) were added to each well and the plates were shaken for 5 min and then left to elute overnight at 4°C. The plates were shaken for 2 min before testing the eluate. A 1:5 dilution of each eluate was made in TPHA buffer (Fuiirebio, Tapan) and 25 l.tL were transferred to a V-well’micro&e plate (Dvnatech, USA). The TPPA test particles were reconstituted according to the manufacturer’s instructions and subsequently diluted 1: 10 with TPHA buffer immediately before use; 25 & of diluted test particles were added to each well (including kit controls, which were not prediluted). The plates were shaken briefly for 10 s and incubated for 90 min at room temperature on a flat, vibration-free surface. Plates were visually evaluated on a lightbox: a negative result was shown by the formation of a tight discrete button, whilst positive results were indicated by clumps of particles with agglutinated edges. In order to assess the sensitivity and specificity of the TPPA, we tested sera with the Serodia Tl?PAa test, according to the manufacturer’s instructions, and compared blood spots and serological results. Among the 1037 samples, 59 were positive by TPPA when using sera and 58 were TPPA positive when using the blood spots. Thus the modified blood spot TPPA test had a sensitivity of 98.3%. None of the positive TPPA blood spot samples was found to be negative by serological testing, giving the modified blood spot TPPA technique a specifity of 100%. The very high sensitivity and specificity of the blood suet TPPA test. comnared to the use of serum. make it acceptable for use as a specific test for T. pallidurn antibodies. Our modification of the existing test for use with blood spots is simple and does not complicate the test procedure. Blood spots can be obtained by finger-prick and the samples can be stored at room temperature, thereby reducing collection, storage and transportation problems.The modified blood spot TPPA test is more economical than most non-treponemal tests (LO. 10 per sample compared with LO.40 per sample for an RPR test). It has the additional advantages of being more sensitive and specific than non-treponemal tests, reducing the risk of false results, and it is easy to perform. The test is therefore appropriate for use in low resource settings, with limited laboratory facilities, for either routine clinical screening or large epidemiological surveys for syphilis. I
I
I
Larsen, S. A., Steiner, B. M. & Rudolph, A. H. (1995). Laboratory diagnosis and interpretation of tests for syphilis.CZinical Microbiolopv Reviews,8, l-2 1. Stevens, R., Pa&, K., Fuller; S., Wiznia, A., Noble, L., Duva, S. &Neal, M. (1992). Blood spot screening and confirmatory tests for syphilis antibody. Journal of Clinical Microbiology, 30,2353-2358.
Received 3July 1997; acceptedforpublication
11 July 1997