Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

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Biologicals 37 (2009) 245e251 www.elsevier.com/locate/biologicals

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies Peter Rigsby a, Catherine Ison b, Matthew Brierley a, Ron Ballard c, Hans-Jochen Hagedorn d, David A. Lewis e, Daan W. Notermans f, Jørn Riis g, Peter Robertson h, Ilkka J.T. Seppa¨la¨ i, Sjoerd Rijpkema j,* a

Biostatistics Section, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, United Kingdom b Sexually Transmitted Bacteria Reference Laboratory, Health Protection Agency, Centre for Infections, Colindale, United Kingdom c Laboratory Reference and Research Branch, Division of STD Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA d Labor Dr. Krone and Partner, Bad Salzuflen, Germany e Sexually Transmitted Infections Reference Centre, National Institute for Communicable Diseases (NHLS), Johannesburg, South Africa f Diagnostic Laboratory for Infectious Diseases & Perinatal Screening Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands g Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark h SEALS Area Serology Laboratory, Prince of Wales Hospital, Randwick, New South Wales, Australia i HUSLAB, Unit of Immunology, Clinical Microbiology, Helsinki University Hospital, Helsinki, Finland j Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, United Kingdom Received 9 December 2008; revised 11 February 2009; accepted 19 March 2009

Abstract A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/ 132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidumspecific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132. Ó 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. Keywords: Syphilis; Serodiagnosis; Antibodies; International standard preparations

1. Introduction Syphilis is a sexually transmitted disease (STD) caused by spirochetes of the species Treponema pallidum subsp.

* Corresponding author. Tel.: þ44 (0) 1707 641000; fax: þ44 (0) 1707 641054. E-mail address: [email protected] (S. Rijpkema).

pallidum. Recent reports show that the incidence of syphilis has risen in both developed and developing countries [1e5]. In Europe and USA, the rise in syphilis has largely been attributed to an increase in unsafe sexual behaviour among men who have sex with men [1,3], while in Africa and Russia, the disease is usually heterosexually transmitted. Syphilis is often associated with HIV infection and enhances its transmission. Untreated congenital syphilis remains a major cause of perinatal morbidity and mortality [3e6]. Therefore worldwide,

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syphilis remains a significant public health problem, which has additional implications for the safety of blood products and organ donations akin to blood borne viruses. Establishment of a rapid and accurate diagnosis remains an essential element in the prevention and treatment of syphilis. Reference laboratories, diagnostic laboratories and test manufacturers need international standards (ISs) to calibrate immunoassays. A recent study by Muller et al. highlighted the importance of standardisation and proficiency testing for the maintenance of high standards in syphilis serology [7]. HS, the 1st IS for syphilitic serum antibodies, was established under auspices of the WHO in 1957 and has a unitage of 24.4 IU ml1 (49 IU per ampoule [8]). The unitage was assigned on the basis of the reactivity in the cardio-lipin and Kahn assays, which were used routinely at that time and preceded the current Venereal Disease Research Laboratory test (VDRL) and the rapid plasma reagin test (RPR). Stocks of HS are now exhausted and a new preparation was needed. A number of serological tests are available to confirm a clinical diagnosis or screen for asymptomatic infection. Tests can be divided in those assays that measure treponema-specific antibodies, such as: the T. pallidum passive particle agglutination assay (TPPA), the fluorescent Treponema antibody (FTA) assay and enzyme immunoassays (EIAs) based on native or recombinant antigens; and the VDRL and the RPR, non-treponemal assays which measure non-specific antibodies. The two latter assays, which are relatively inexpensive, have historically been used as screening tests and can be used to monitor the efficacy of treatment. In contrast, the specific treponemal tests are more expensive but significantly more specific than the non-treponemal tests. Historically, they were used only as confirmatory assays; however with the advent of automation, some tests (primarily EIAs) have been used as screening assays. However, this practice should only be adopted in low prevalence settings since samples remain positive even after successful therapy [9]. We selected two human plasma pools (05/122 and 05/132) as candidates to replace HS and designed a collaborative study to assign a unitage to these preparations. Preliminary analysis revealed that 05/122 contains specific IgG but not IgM and is not reactive in the cardio-lipin based tests and that 05/132 contains specific IgG and IgM and is strongly reactive in the RPR test (Table 1). Therefore, 05/122 and 05/132 can be taken to represent the antibody response of patients with previously treated early syphilis and active syphilis respectively [9]. Participating laboratories of the collaborative study were asked to analyse the presence of syphilitic antibodies in 05/ 122 and 05/132 by TPPA and VDRL or RPR. In addition participating laboratories were encouraged to use additional assays that are part of their diagnostic routine. Our primary aims were to: 1) Assess the suitability of the two freeze-dried preparations 05/122 and 05/132 as candidate ISs for TPPA and RPR/ VDRL assays and calibrate these in terms of HS reactivity in the TPPA and RPR/VDRL.

Table 1 Samples used in this study. Study NIBSC Sample code code description

RPR TPPA

HS D,F

64 8

>1280 pos 1280 pos

pos pos

neg

1280

pos

neg

neg neg neg neg

neg 1280 neg neg

neg pos neg neg

neg pos neg neg

A E Ca B G a

HS 1st international standard 05/132 Candidate international standard (pool of 25) 05/122 Candidate international standard (pool of 3) 97/682 Normal serum from 1 donor 05/142 Positive serum from 1 donor 83/571 Normal serum from 1 donor 82/585 Normal serum from 1 donor

EIA Total Ig IgM

Tested prior to freeze drying.

2) To assess the reactivity of the two candidate ISs and HS in various immunoassays currently in use. 2. Materials and methods 2.1. Participating laboratories Eight laboratories from eight countries, including national reference laboratories, tested the samples and supplied test data (see author list for details). The participating laboratories were recruited from a network of diagnostic reference laboratories for STDs maintained by one of the authors (CI). National blood banks or manufacturers of diagnostic assays were not included in this study. Data were collected and analysed at the National Institute for Biological Standards and Control (NIBSC), United Kingdom. Throughout the study, participating laboratories have been identified by a randomly assigned code number to maintain confidentiality. 2.2. Samples Each participating laboratory received two sets of samples comprising six coded ampoules and one ampoule of HS. The study codes, NIBSC codes, the reactivity of the sample in RPR, TPPA, EIA and a brief characterisation are given in Table 1. All samples tested negative for antibodies to HIV 1/2 and Hepatitis C, and Hepatitis B surface antigen. Sample 05/ 142 was obtained from the Regional Blood Transfusion Service (Leeds, UK) and permission for use was obtained at source. Duplicates of sample 05/132 were included in the sample set to provide an independent measure of withinlaboratory variability. Processing of candidate ISs and their use were approved by the local Human Materials Advisory Committee (05/038 SR and 05/039 SR). All samples were distributed as lyophilized preparations at room temperature by courier. Samples that were used to analyse the stability of 05/ 132 and 05/122 were distributed on dry ice. 2.3. Characterisation of the proposed international standard 05/122 Three samples (average volume: 281 ml) were obtained from the Regional Blood Transfusion Service and permission

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Table 2 Assays used in this study. Test format

Name of test

Manufacturer

Titration assay

Rapid plasma reagin test

Axis Shield Diagnostics Ltd., BD-Macro-Vue, Biokit, Wampole Impact, In-house Oxoid, Murex BioMe´rieux/DIFCO, Zeus/BAG BioMe´rieux/DIFCO, Zeus/BAG Fujirebio Serodia-TP. PA Abbott, Microgen bioproducts Ltd, Mikrogen Gmbh, Newmarket Laboratories, Radim SpA Virotech, In-house

Immunoassay

Venereal disease research laboratory test Fluorescent treponema antibody (FTA) assay 19S IgM FTA T. pallidum passive particle agglutination test Enzyme Immuno Assay Ig, IgG, IgM Immunoblot IgM, IgG

for use was obtained at source. Samples tested positive by EIA (Newmarket Laboratories, Newmarket, UK) and were negative in the RPR (Axis Shield Diagnostics Ltd Cambridge, UK) and IgM Capture EIA (Microgen Bioproducts Ltd, Camberley, UK). Samples were stored at 20  C. At NIBSC, samples were thawed, pooled and dispensed in 1 ml aliquots into glass ampoules coded 05/122. The pool was positive by EIA, negative in the RPR and negative in the IgM capture EIA (Table 1). The mean fill weight for 13 ampoules was 1.0096 g with coefficient of variation (CV) of 0.34%. On the same day, freeze-drying under vacuum was started and completed after four days. Ampoules were back filled with pure N2 (moisture content < 10 ppm). Residual moisture measured by the KarlFischer method for 6 ampoules was 0.4066 % (CV of 9.44%). Twenty ampoules were rejected during the production process, 20 ampoules were held for accelerated degradation studies and 665 ampoules were stored at 20  C. These are available for distribution by NIBSC. 2.4. Characterisation of the proposed international standard 05/132 One litre of pooled human plasma was collected in South Africa from 25 donors with active syphilis and for this purpose local ethical permission was obtained (HD Supplies, Aylesbury, UK). The pool tested positive by the TPPA, EIA, RPR and IgM Capture EIA tests (Table 1). At NIBSC, samples were thawed and dispensed in 1 ml aliquots into glass ampoules coded 05/132. The mean fill weight for 12 ampoules was 1.0069 g (CV of 0.17%). On the same day, freeze-drying under vacuum was started and completed after four days. Ampoules were back filled with pure N2 (moisture content < 10 ppm). Residual moisture measured by the Karl-Fischer method for 6 ampoules was 0.6941% (CV of 26.09%). Twelve ampoules were rejected during the production process, 20 ampoules were held for accelerated degradation studies and 929 ampoules were stored at 20  C. These are available for distribution by NIBSC. 2.5. Diagnostic assays The assays used in this study are summarised in Table 2. Two types of assay were distinguished: titration methods and EIAs.

Laboratory codes

Total

1,2,3,4,5,8

6

6,7,8 2,6,7 2,6,7 1,2,4,5,6,7,8 1,2,3,5,7,8

3 3 3 7 6

2,7

2

Titration methods yield an endpoint titre and participating laboratories that use this type of assay were requested to test each sample in duplicate and to test the different ampoule sets on separate days. Since EIAs produce a numerical response such as an absorbance or a fluorescence value, participating laboratories were requested to report the results of four sequential doubling dilutions (e.g. 1/2, 1/4, 1/8 and 1/16) for each sample. Participating laboratories using in-house tests followed standard procedures, while laboratories that used commercially available tests followed procedures described by the manufacturer. Descriptions of the procedures were returned to NIBSC accompanied by the raw data for analysis. 2.6. Data analysis For the titration methods, relative endpoint titres were expressed as potencies relative to HS. For the EIA methods, assays were analysed using the principles of parallel-line bioassay comparing transformed assay response to log Table 3 Results of RPR and VDRL expressed as average endpoint dilutions. Lab

Method

1

RPR

2

RPR

3

RPR

4

RPR

5

RPR

6

VDRL

7

VDRL

8

RPR

8

VDRL

Set

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

Geometric Mean 95% Confidence Interval % GCV

Endpoint dilutions HS

D

F

64 64 54 53.8 256 128 45.3 64 32 32 90.5 90.5 64 64 64 64 32 32

8 8 4 3.4 32 13.5 16 16 2.8 2.8 9.5 11.3 5.7 8 8 8 4 4

8 8 2 4 45.3 8 16 16 4 4 13.5 11.3 5.7 5.7 16 16 8 8

Geometric mean (D & F)

8.0 3.2 19.9 16.0 3.4 11.3 6.2 11.3 5.7 7.9 (4.8e13.0) 90

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Table 4 Results of RPR and VDRL expressed as relative potencies. Lab

Method

Set

Potency (IU ml1 calculated relative to HS) D

F

1

RPR

2

RPR

3

RPR

4

RPR

5

RPR

6

VDRL

7

VDRL

8

RPR

8

VDRL

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

3.05 3.05 1.81 1.54 3.05 2.57 8.62 6.10 2.14 2.14 2.56 3.05 2.17 3.05 3.05 3.05 3.05 3.05

3.05 3.05 0.90 1.81 4.32 1.53 8.62 6.10 3.05 3.05 3.64 3.05 2.17 2.17 6.10 6.10 6.10 6.10

Geometric Mean 95% Confidence Interval % GCV

concentration [10]. All mean potencies shown in this report are unweighted geometric mean potencies. Variability between estimates is shown using geometric coefficients of variation (GCV). 3. Results and discussion 3.1. RPR and VDRL results Eight laboratories reported positive non-treponemal test results for samples HS, D and F (05/132, see Table 1 for details). All other samples were reported as negative (see Table 3). One laboratory performed both RPR and VDRL tests. Positive results are summarised as endpoint dilutions and as potencies relative to HS in Tables 3 and 4. For samples D and F, a geometric mean potency of 3.14 IU ml1 (95% confidence limits: 2.22e4.44; n ¼ 9) is calculated relative to HS (24.4 IU ml1, [8]). Laboratory 8 obtained identical relative potencies in RPR and VDRL for the same set of ampoules. Inclusion of only one set of results from this participant in the calculation of the mean gives 3.02 IU ml1 for 05/132 (95% confidence limits: 2.04e4.46; n ¼ 8). No significant difference between RPR and VDRL potencies was detected and no laboratories were found to be outliers. Higher variability is noted for the endpoint dilutions (GCV 90%; n ¼ 9) compared to the relative potencies (GCV 57%; n ¼ 9), indicating that use of a common standard improves agreement between laboratories. Variability within laboratories has been assessed using the potencies of the coded duplicates of 05/132 (D and F) relative to one another. All potencies were between 0.5 and 2.0 and the geometric mean of

Geometric mean

% GCV

Potency of F relative to D

3.05

0

1.46

39

2.68

54

7.25

22

2.55

22

3.05

15

2.37

19

4.31

49

4.31

49

1.00 1.00 0.57 1.17 1.38 0.55 1.00 1.00 1.44 1.44 1.36 1.00 1.00 0.69 2.00 2.00 2.00 2.00 1.17 (0.95e1.43) 50

3.14 (2.22e4.44) 57

1.09 shows agreement with its expected value. The GCV of 50% indicates a comparable level of variability to that observed between laboratories. The unitage of HS was originally defined using cardio-lipin antigen-based assays such as the VDRL assay [8]. The reactivity of 05/132 falls within the range of the RPR/VDRL and this preparation can be used as a reference standard in these assays. Relative to HS, 05/132 can be assigned a unitage of 3 IU per ampoule. 3.2. TPPA results Seven laboratories reported positive results for samples HS, A, C, D and F (see Table 5). All other samples were reported Table 5 Results of TPPA expressed as geometric mean endpoint dilutions. Lab

1 2 4 5 6 7 8

Set

1 2 1 2 1 2 1 2 1 2 1 2 1 2

Endpoint dilutions HS

A

C

D

F

163840 >28963 >20480 >28963 4305 3620 >1280 >1280 24355 28963 >26908 36204 20480 20480

1280 1810 1280 1280 320 190 1280 1280 1810 1522 761 640 587 761

5120 3620 3948 3320 453 1076 >1280 >1280 6089 5120 2862 3200 1174 1396

14481 4482 12177 13280 1280 3044 >1280 >1280 14482 14482 5724 6400 10240 10240

28963 >14482 7241 13280 905 2560 >1280 >1280 14482 10240 5724 6400 2560 3620

P. Rigsby et al. / Biologicals 37 (2009) 245e251

249

Table 6 Results of TPPA expressed as relative potencies. Lab

Set

1

1 2 2 1 2 4 1 2 6 1 2 7 1 2 8 1 2 Geometric Mean 95% Confidence Interval % GCV

Potency relative to 05/132 (D & F)

Potency relative to A

Individual results

Geometric mean

Individual results

Geometric mean

A

C

A

C

C

C

0.06 e 0.14 0.10 0.30 0.07 0.12 0.12 0.13 0.10 0.11 0.12

0.25 e 0.42 0.25 0.42 0.39 0.42 0.42 0.50 0.50 0.23 0.23

0.06

0.25

2.83

0.11

0.32

0.14

0.40

0.12

0.42

0.11

0.50

0.12

0.23

4.00 2.00 3.08 2.59 1.42 5.66 3.36 3.36 3.76 5.00 2.00 1.83

0.11 (0.08e0.15) 34

0.34 (0.25e0.47) 36

as negative. One participating laboratory did not perform the TPPA. Results are summarised as endpoint dilutions. Results of participant 5 were excluded from further calculations as endpoint dilutions were obtained for sample A only. Laboratories 1, 2 and 7 also obtained endpoint dilutions for HS that were outside of the assay range used. As a result of this, the relative potency of 05/122 (A) could only be compared to 05/132 and not to HS (see Table 6). Taking the potency of 05/ 132 to be 3 IU ml1, the potency of 05/122 in the TPPA relative to 05/132 was calculated as 0.33 IU ml1 (95% confidence limits: 0.24e0.45; n ¼ 6). The GCV of 34% demonstrates good agreement between laboratories. In a recent study, Manavi et al. showed that TPPA is the most sensitive assay for the detection of primary syphilis and was superior to RPR/VDRL [11]. Thus an IS with relatively low antibody potency is better suited for the TPPA. The reactivity of 05/132 and 05/122 falls within the range of the TPPA and both these preparations can be used as reference standards in this assay. Relative to 05/132, 05/122 can be assigned a unitage of 300 mIU per ampoule.

2.83 2.83 3.36 4.34 1.92 2.93 (2.21e3.88) 31

TPPA. Some loss of activity was seen at þ37  C, but this falls within the experimental error of the procedure and no loss was seen at þ4  C or þ20  C. Therefore no predicted loss of activity can be calculated (see Tables 7 and 8). Our findings strongly suggest that the unitage of 05/132 and 05/122 will remain stable at 20  C. 3.4. Recommendation by the Expert Committee on Biological Standardization In October 2007, the WHO Expert Committee on Biological Standardization (ECBS) recommended that, on the basis of the results from the RPR and VDRL assays, 05/132 is established as the 1st IS for human syphilitic plasma IgG and IgM with an assigned potency of 3 IU per ampoule relative to HS. The ECBS also recommended that on the basis of the results from TPPA assays, 05/122 is established as the 1st IS for human syphilitic plasma IgG with an assigned potency of 300 mIU per ampoule relative to 05/132 [13]. 3.5. Ig(G) detection by EIA and FTA

3.3. Stability studies Following storage at various temperatures for one year, 05/ 132 and 05/122 were tested in one laboratory in the RPR and

EIA and FTA results are summarised in Tables 9 and 10 respectively. In most cases, the range of EIA responses for samples HS, D and F did not overlap with the range observed for

Table 7 Results from RPR for 05/122 and 05/132 stored at elevated temperatures for one year. Sample

05/122

05/132

Storage temperature 20  C þ4  C þ20  C þ37  C 20  C þ4  C þ20  C þ37  C

Assay 1 endpoint dilution Duplicate 1

Duplicate 2

Geometric mean

Assay 2 endpoint dilution Duplicate 1

Duplicate 2

Geometric mean

Geometric mean potency relative to 20  C

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

NEG NEG NEG NEG 1:8 1:8 1:8 1:4

e e e e e 1.00 1.00 0.50

P. Rigsby et al. / Biologicals 37 (2009) 245e251

250

Table 8 Results from TPPA for 05/122 and 05/132 stored at elevated temperatures for one year. Sample

Storage temperature

05/122

20  C þ4  C þ20  C þ37  C 20  C þ4  C þ20  C þ37  C

05/132

Assay 1 endpoint dilution

Assay 2 endpoint dilution

Duplicate 1

Duplicate 2

Geometric mean

Duplicate 1

Duplicate 2

Geometric mean

Geometric mean potency relative to 20  C

1:640 1:640 1:640 1:320 1:5120 1:5120 1:5120 1:5120

1:640 1:640 1:640 1:320 1:5120 1:5120 1:5120 1:5120

1:640 1:640 1:640 1:320 1:5120 1:5120 1:5120 1:5120

1:640 1:640 1:640 1:320 1:5120 1:5120 1:5120 1:2560

1:640 1:640 1:640 1:640 1:5120 1:5120 1:5120 1:2560

1:640 1:640 1:640 1:453 1:5120 1:5120 1:5120 1:2560

e 1.00 1.00 0.59 e 1.00 1.00 0.71

Table 9 Results of Ig(G) EIAs expressed as relative potencies. Lab

Method

Potency (IU ml1 calculated relative to HS)

Set

1

Ig EIA

2

IgG EIA

3

IgG EIA

5

Ig EIA

1 2 1 1 2 2 1 1 2 2 1 1 2 2

Geometric mean

D

F

11.22 9.76 8.30 8.78 7.08 Non-parallel 14.15 HS non-linear 9.27 D non-linear 9.03 8.78 9.03 8.30

8.78 8.05 9.03 9.03 7.81 Non-parallel 10.49 HS non-linear 9.27 15.13 11.22 8.54 10.98 8.54

9.27 8.30

11.47

9.27

Geometric Mean

Potency of C relative to A

Geometric mean

0.31 0.30 C non-linear C non-linear 0.71 0.72 0.34 0.44 0.31 0.40 0.48 0.44 0.48 0.46

0.30 0.71

0.37

0.47

9.52

A and C. Therefore, as a common response range is required for parallel-line analysis, potencies of D and F relative to HS have been calculated separately from potencies of C relative to A. FTA results were obtained from three laboratories and were broadly in agreement with EIA results (see Table 10). Two participating laboratories reported qualitative results for EIA and FTA. HS, A, C, D and F were identified as positive (results not shown). We conclude that the reactivity of 05/132 and 05/ 122 fall within the range of the Ig(G) EIAs and FTAs and these preparations can be used as a positive control in these assays.

0.44

3.6. Detection of IgM by EIA and 19S IgM FTA FTA with fractionated serum identified IgM correctly in 05/132 (D & F) and HS. Samples B, C, E and G were reported as negative and a false positive result was reported for 05/122 (A; Table 10). The IgM capture ELISA detected all IgM positive samples and returned both a positive and a negative result for duplicates of sample C (results not shown). Thus IgM capture EIA confirmed the results of the 19S IgM FTA. It should be noted that sample C was

Table 10 Results of the FTA. Lab

Set

2

1 2 1 2 1/2 1/2

6 7b

Endpoint dilution or titre for Ig(G) FTA

Endpoint dilution or titre for 19 S IgM FTA

HS

A

C

D

F

HS

A

C

D

F

3520a 2560a þþ þþ þþþa þþþþ

160a 160a þþ þþ þa þþþþ

80a 80a þ þ þa þ

960a 640a þþ þþ þþþa þþþ

880a 720a þþ þþ þþþa þþþþ

50 55 þ þ þþþþ ND

NEG NEG NEG NEG þþ ND

NEG NEG NEG NEG NEG ND

12 12 þ þ þþþ ND

13 13 ND ND þþ ND

Preparations B, E and G were reported as negative. a IgG. b Only one sample of set 1 or 2 was tested.

P. Rigsby et al. / Biologicals 37 (2009) 245e251

unequivocally positive by the IgM capture EIA prior to freeze drying (Table 1); therefore we conclude that anti-treponemal IgM may not be stable after freeze drying and as a result, low levels of specific IgM may decrease beyond detection. All coded samples were reported negative by indirect IgM EIA (results not shown). In the current study, the sensitivity of the 19S IgM FTA was comparable to IgM capture EIA and superior to indirect IgM EIA. This observation is in line with findings by Lefevre et al. who reported that for primary syphilis the diagnostic sensitivity of 19S IgM FTA and the capture IgM EIA are similar [12]. One laboratory performed blotting and identified IgM in HS, D and F and one laboratory detected IgM detection by TPPA and reported HS and D, but not F, as positive (results not shown). We conclude that 05/132 is reactive in the 19S IgM FTA, IgM capture EIA and IgM blot, hence 05/132 can be used as a positive control in these assays. A unitage for the IgM content of 05/132 was not assigned due to the paucity of data. We did not seek recommendations from the ECBS in this respect. Acknowledgements We are grateful to Ms S. Coughlan (Centre for Biological Reference Materials, NIBSC) for coding and packaging of samples and organising their distribution. The technical assistance from the following staff members is greatly appreciated: Ms B. Marsden and H. Patel (Sexually Transmitted Bacteria Reference Laboratory, Health Protection Agency Centre for Infections); Ms S. Kikkert and P. Thompson (WHO Reference Center for Syphilis Serology, Centers for Disease Control and Prevention); Ms S. Jorde, B. Hartmeier and I. Pieper (Labor Dr. Krone and Partner); Mr S. Khumalo, Dr I. Zietsman and Mr. F. Radebe (STI Reference Centre, NICD, NHLS); Mr M. Mommers (Laboratory for Infectious Diseases & Perinatal Screening, Centre for Infectious Disease Control, RIVM); Scientific and technical staff (South Eastern Sydney Area

251

[SEALS] Serology laboratory); Ms M. Korpinen, A. Riutta and R. Va¨isa¨nen (HUSLAB, Helsinki University Hospital).

References [1] Centers for Disease Control and Prevention. Primary and secondary syphilis e United States, 2003e2004. MMWR 2006;55:269e73. [2] Chen ZQ, Zhang GC, Gong XD, Lin C, Gao X, Liang GJ, et al. Syphilis in China: results of a national surveillance programme. Lancet 2007;369:132e8. [3] Nicoll A, Hamers FF. Are trends in HIV, gonorrhoea, and syphilis worsening in Western Europe? BMJ 2002;324:1324e7. [4] Reynolds SJ, Risbud AR, Shepherd ME, Rompalo AM, Ghate MV, Godbole SV, et al. High rates of syphilis among STI patients are contributing to the spread of HIV-1 in India. Sex Transm Infect 2006;82:121e6. [5] Salakhov E, Tikhonova L, Southwick K, Shakarishvili A, Ryan C, Hillis S. Congenital syphilis in Russia: the value of counting epidemiologic cases and clinical cases. Sex Transm Dis 2004;31:127e32. [6] Mullick S, Watson-Jones D, Beksinska M, Mabey D. Sexually transmitted infections in pregnancy: prevalence, impact on pregnancy outcomes, and approach to treatment in developing countries. Sex Transm Infect 2005;81:294e302. [7] Muller I, Brade V, Hagedorn HJ, Straube E, Schorner C, Frosch M, et al. Is serological testing a reliable tool in laboratory diagnosis of syphilis? Meta-analysis of eight external quality control surveys performed by the german infection serology proficiency testing program. J Clin Microbiol 2006;44:1335e41. [8] Weiss-Bentzon M, Krag P. The international standard for human syphilitic serum. Bull WHO 1961;24:257e64. [9] Egglestone SI, Turner AJ. Serological diagnosis of syphilis. Commun Dis Public Health 2000;3:158e62. [10] Finney DJ. Statistical method in biological assay. 3rd ed. London: Charles Griffin; 1978. [11] Manavi K, Young H, McMillan A. The sensitivity of syphilis assays in detecting different stages of early syphilis. Int J STD AIDS 2006;17:768e71. [12] Lefevre JC, Bertrand MA, Bauriaud R. Evaluation of the Captia enzyme immunoassays for detection of immunoglobulins G and M to Treponema pallidum in syphilis. J Clin Microbiol 1990;28:1704e7. [13] WHO/BS/07.2059. Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies. http://www.who.int/biologicals/expert_committee/ BS%202059%20syphilitic%20antibodies.pdf.