WHO/BS/2011.2181 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17 to 21 October 2011
Evaluation of two International Reference Standards for antibodies to
Trypanosoma cruzi in a WHO collaborative studyMarcia Otani1, Jason Hockley2, Carmen Guzmán Bracho3, Sjoerd Rijpkema4*, Alejandro O.
Luquetti5, Robert Duncan6, Peter Rigsby2, Pedro Albajar-Viñas
7
, Ana Padilla
8
1Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brasil; 2Biostatistics Section, National Institute for Biological Standards and Control (NIBSC), Potters Bar, United Kingdom (UK); 3Departamento de Parasitología, Instituto de Diagnóstico y Referencia Epidemiológicos, Secretaria de Salud, Mexico D. F., Mexico; 4Division of Bacteriology, NIBSC, UK; 5Laboratory for Research on Chagas disease, Hospital das Clínicas, Universidade Federal de Goiás, Goiânia, Brazil;
6
Division of Emerging and Transfusion Transmitted Disease, Center for Biologics Evaluation and Research, US FDA;
7
Chagas disease Programme, Control of Neglected Tropical Diseases and;
8
Blood Products and related Biologicals, Quality Assurance and Safety: Medicines, World Health Organization, Geneva, Switzerland; *(Corresponding author).
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Summary. Two freeze dried preparations of defibrinated human plasma containing anti-Trypanosoma cruzi antibodies, coded 09/186 and 09/188, were assessed for their suitability as International Reference Standards (IS) for the serodiagnosis of Chagas disease. A WHO collaborative study was undertaken by 24 laboratories from 16 countries. Preparations 09/186 and 09/188 were tested in 30 commercially available assays: enzyme immunoassays (EIA, n=16), one chemiluminescent immunoassay, indirect immunofluorescence assays (IFA, n=4), indirect hemagglutination assays (IHA, n=4), one particle agglutination assay and rapid immunochromatographic assays (RIC, n=4). In addition 10 in-house assays were used: EIA (n=2), IFA (n=5) and western blots (n=3). A radioimmunoprecipitation assay (RIPA) was used to characterize prototypes of 09/186 and 09/188 in a pilot study.Both preparations 09/186 and 09/188 were identified as sero-positive in all assays except for one laboratory. Based on the results of the collaborative study, the reactivity in EIA and ChLIA was used as a guide to establish the unitage mL-1. The geometric mean endpoint titre estimated ranged from 2 to 37 for preparation 09/186, and from 3 to 59 for preparation 09/188. For 09/186, the overall Geometric mean (GM) was calculated as: 11 (Geometric Coefficients of variation [GCV] 104.7%;
n=29) for assays using native antigen and 9 (GCV 89.4%; n=25) for assays using recombinant antigen.
For 09/188, the overall GM was calculated as: 17 (GCV 101.3%; n=29) for assays using native antigen and 16 (GCV 125.1%; n=25) for assays using recombinant antigen.
It is proposed that preparations 09/186 and 09/188 are established as the 1st WHO IS for anti- Trypanosoma cruzi antibodies:
- Preparation09/186, defined as the anti-T.cruzi antibody Standard representative of the region where T.cruzi II is predominant, with an arbitrary unitage of 1 TcII International Unit mL-1 for the undiluted concentration after reconstitution, according to the instructions for use.
- Preparation 09/188, defined as the anti-T.cruzi antibody Standard representative of the region where T.cruzi I is predominant, with an arbitrary unitage of 1 TcI International Unit mL-1 for the undiluted concentration after reconstitution, according to the instructions for use.
These standards , containing 0.5 mL freeze dried plasma, will be distributed together, with an assigned unitage of 0.5 IU per ampoule. The unitage to be associated with the endpoint titre can thus be taken as the reciprocal value of the titre, and this will inform the user of the relative strength of 09/186 and 09/188 in the assay of use.
INTRODUCTION
Chagas disease (Human American trypanosomiasis) is caused by an infection with the protozoan parasite Trypanosoma cruzi and was first described in 1909 (1, 2). It is estimated that around 10 million people are currently infected by T. cruzi in the world, and more than 10,000 people die from chronic clinical manifestations every year, mainly Chagasic cardiopathy. The endemic area of the disease is Latin America, from Mexico in the north to Argentina and Chile in the south. Nevertheless, during the last twenty years, implementation of large-scale vector control programmes in Central and South American countries, together with mandatory screening of blood donations for anti-T. cruzi antibodies, have resulted in an important reduction in the incidence and prevalence of the disease. On the other hand, in the past decades, mainly due to population mobility, Chagas disease cases have been increasingly detected in disease non-endemic countries in the Region of the Americas (Canada and the United States of America), the Western Pacific Region (mainly Australia and Japan) and the European Region (with reported cases in 16 countries) (3, 4).
The Chagas disease vector, a haematophagous triatomine bug, principally found in the Americas, is infected when it sucks blood from infected mammals, including humans. The parasite multiplies in the hindgut of the insect and divides into metacyclic trypomastigotes, a highly infectious form that is transmitted when humans put in contact the contaminated faeces of the vector with any skin break, including the bite, or the eye or oral mucous membranes. In disease-endemic, but also in non-endemic areas, the parasite can be transmitted by ingestion of contaminated food, blood transfusion, congenital transmission, organ transplantation or laboratory accident (5). Chagas disease starts with an acute phase, which lasts for 6–8 weeks, and it follows with a chronic phase that persists for life without successful anti-parasitic treatment and, consequently, without parasitological cure. In most of the acute cases symptoms are absent or mild but can include fever, headache, enlarged lymph glands, pallor, muscle pain, difficulty in breathing, swelling and abdominal or chest pain. In less than 50% of people bitten by a triatomine bug, first visible characteristic signs can be a skin lesion or a purplish swelling of the lids of one eye. During the chronic phase, the parasites are hidden mainly in the heart and digestive muscle. Up to 30% of patients suffer from cardiac disorders and up to 10% suffer from digestive (typically enlargement of the oesophagus or colon), neurological or mixed alterations. In later years the infection can lead to sudden death or heart failure caused by progressive destruction of the heart muscle.
Laboratory diagnosis of the acute phase is based on the microscopic detection of the trypomastigote form of the parasite. Two to four months after the acute phase, most infected individuals enter the chronic phase with no or limited parasitemia, hence the laboratory diagnosis relies on the detection of IgG antibodies against T. cruzi (6). The application of biochemical and molecular techniques has revealed great genetic diversity among T. cruzi isolates and two major groups have been identified: T.
cruzi I (TcI) and T. cruzi II (TcII) (7, 8). TcI circulates in domestic and sylvatic cycles and is predominantly found north of the Amazon basin (9, 10, 11, 12, 13) and TcII is associated with the domestic cycle and is predominant in Southern Latin American countries (14, 15, 16, 17, 18, 19, 20). In fact, since the first publication on the subject by Carlos Chagas in 1924 (21), several studies have described regional differences both in terms of parasitological, serological and clinical characteristics, together with anti-parasitological treatment response (5, 22, 23, 24). However, confirmation of the T.
cruzi genotype from infected donors in the chronic phase remains difficult as parasitaemia is usually very low or absent.
Available assay methods for serodiagnosis of Chagas disease are the enzyme immunoassay (EIA) (25), the indirect immunofluorescence assay (IFA) (26, 27), the indirect haemagglutination assay (IHA) and particle agglutination assay (PAG) (28), chemiluminescent immunoassay (ChLIA) (29), and rapid immunochromatographic assay (RIC) (30). Western blot (31) and RIPA (32, 33) are used as supplemental assays. These tests usually employ a mix of antigens, mainly obtained from the epimastigote form of the parasite, but also from the trypomastigote and amastigote forms, either as a lysate, purified proteins, recombinant antigens or synthetic peptides (34). Most commercial tests have been developed with antigens of parasite forms of T. cruzi II, in South America.
The first “WHO Consultation on International Biological Reference Preparations for Chagas Diagnostic Tests” was convened by the WHO Programme on Blood Products and Related Biologicals/Quality and Safety: Medicines Team, in July 2007 (18). The objective of the Consultation was to discuss the type of materials needed to support the development of global reference standards for use in the quality control of Chagas disease diagnostic tests, based on the detection of antibodies to T.cruzi. During the consultation, the development of International Biological Reference Preparations for antibodies against T. cruzi was agreed using samples obtained from disease endemic countries, one from the region where T. cruzi I prevails (i.e. Mexico), and another one from the region where T. cruzi II prevails (i.e. Brazil). It was considered that the availability of internationally agreed reference preparations would contribute to facilitating the control of the analytical sensitivity of in-house and commercially available tests by manufacturers, regulators, blood establishments, reference and diagnostic laboratories. Furthermore, it would facilitate development of new tests and support harmonization of international regulations.
The suitability of materials selected for the production of two reference standards derived from plasma obtained from healthy blood donors, positive for T. cruzi, living in areas where Chagas disease is endemic and who are at a high risk of coming into contact with triatomine insects was discussed at the second "WHO Consultation on International Biological Reference Preparations for Chagas Diagnostic Tests", in January 2009. The composition of the global reference standards, intended use and the design of a WHO collaborative study (CS) to calibrate the proposed preparations were also considered. There was also consensus to use samples of medium reactivity, suitable for the various immunoassay formats.
The CS was designed to validate and characterize the antibody reactivity of the above proposed candidate preparations (subsequently coded 09/186 and 09/188) including both, screening tests and supplementary tests. Efforts were made to ensure that most commercially available serodiagnostic assays for Chagas disease be represented. The primary aims of the CS were to:
1) Characterize the reactivity of the two candidate ISs in IFAs, IHAs, western blots, PAG, RIPA and RIC, currently in use.
2) Assess the suitability of the two freeze-dried preparations 09/186 and 09/188 as candidate International Standards (ISs) for the quality control of EIAs;
The results of the WHO collaborative study are presented in this Report. The list of participants in the collaborative study and at the above Consultations are attached in Appendix 2 and 3.
MATERIALS AND METHODS
Selection of blood donor plasma samples. Plasma samples from blood donors with previous positive serology in anti-T. cruzi antibody detection tests were provided by the Hidalgo State Centre of Blood Transfusion, Mexico, and the Fundação Pró-Sangue Hemocentro de São Paulo, Brazil, for the preparation of the candidate reference standards calibrated in this study. Two positive plasma units were selected at the Hidalgo State Centre from male blood donors (43 and 48 years old). The reactivity of these samples was evaluated by IHA, IFA, EIA and WB at the Instituto de Diagnóstico y Referencia Epidemiológicos, Mexico Federal District. Three positive plasma units were collected at the Fundação Pró-Sangue from blood donors original from the following endemic areas: Minas Gerais, Brazil (female, 31 years old); Canela, Chile (male 58 years old) and Pernambuco, Brazil (female, 51 years old). EIA and IFA tests were used for confirmation of the donors as anti-T.cruzi positive together with evidence of high risk of T. cruzi infection in a questionnaire to assess the epidemiological profile and autochthonous endemic origin. Plasma samples obtained from Mexico were considered to represent the TcI predominant region and plasma samples obtained in Brazil and Chile the TcII predominant region. Due consent was obtained from all blood donors.
Efforts to isolate the parasite from blood donor samples were unsuccessful to date and hence the T. cruzi genotype could not be confirmed. This difficulty to isolate the parasite in hemoculture was expected given the very low or absent parasitemia in the chronic phase of the Chagas disease.
Leishmania chagasi is known to be the most important agent that can cause cross reaction with anti- T.cruzi assays (35, 36). Donor samples and the two seropositive pools of defibrinated human plasma were tested negative by RIC IT-LEISH® (DiaMed).
Preparation and characterization of the candidate bulk materials. All plasma units were
defibrinated at the Fundação Pró-Sangue by adding 0.5 mL of a 200 mM CaCl2 solution to a 100 mL unit of plasma. The mixture was incubated at 37°C for 2 hours and for 24 hours at 4°C and the fibrin clot was removed by centrifugation at 6000 g for 30 min. CaCl2 was removed from the supernatant by dialysis using a cellulose membrane with a cutoff of 12 kDa (Sigma, Germany). The defibrinated plasma was filtered through a 5 µm pore size membrane (Sigma) to remove residual fibrin particles.
As a preservative, Bronidox (5-Bromo-5-nitro-1,3-dioxane, Henkel Chemicals, Germany) was added to a final concentration of 0.05%.
Two seropositive pools of defibrinated human plasma, representative of the latent (chronic) stage of infection with T. cruzi in Brazil and Mexico were prepared. The candidate material representing the TcI predominant region was constituted of 200 mL of defibrinated plasma from each of the two selected Mexican donations and diluted with two units of 200 mL of negative defibrinated plasma each from the TcI predominant region to prepare a total volume of 800 mL. The candidate material representing the TcII predominant region was prepared by mixing 80 mL, 120 mL and 60 mL of defibrinated plasma selected from two Brazilian and one Chilean donation. The 260 mL pool of positive units was diluted in 1,240 mL of negative defibrinated plasma originated from seven seronegative Brazilian donors to obtain a total volume of 1,500 mL.
Both candidate bulk materials (“TcI” and “TcII”) were tested with 3 different EIAs and an IFA for confirmation of positive reactivity for detection of anti-T.cruzi antibodies. In order to know whether lyophilisation could affect antibody concentration titres, a pilot study was also carried out.
Approximately 50 mL of each of the “TcI” and “TcII” bulk materials were freeze-dried (FD) at the National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK, and tested in a pilot study to assure that the antibody concentration in each preparation was in an appropriate range for multiple assay types and multiple laboratories and to ensure the reactivity of the original native liquid materials would remain after freeze drying and reconstitution.
Sets of eight vials (two FD and two liquid for each of the two preparations) were distributed to 5 laboratories: 3 using EIA, IHA and IFA, one using TESA blot and one using RIPA. The results were reported to the NIBSC who analysed the results. The reactivity of the liquid candidate bulk materials is shown in Table 1. The antibody potency of the reconstituted FD preparations relative to liquid prior to FD, shown in Table 2 was characterized by EIA (n=4), IHA (n=3), one PAG and IFA (n=2), which did not reveal loss of potency after FD. The reactivity of the preparations, either in their native liquid form or as reconstituted FD preparations, was also characterized in TESA blot and RIPA. Results are presented in Table 3, and Figs 1 and 2. In TESA blot, freeze-drying resulted in slightly lower band intensities for the TcII bulk material diluted 1:800 - 1:1600 (Fig. 1). Reactivity in TESA blot was observed with bands of a MW of 150-160kDa and with bands of 95 kDa. In TESA blots of the TcI bulk material, freeze drying had no effect on band intensity (Fig. 1). This TcII bulk material was tested in RIPA either in its liquid form or as a reconstituted FD preparation (Fig. 2). Both preparations reacted strongly with the 72 and 90 kDa antigens, even up to 1:100 dilution. Reactivity to these bands is considered to be the primary readout for a positive reaction. Counts per minute (CPM) were also determined prior to PAGE and subsequent autoradiography. The CPM for the liquid preparation and the reconstituted FD preparation is given in Table 3. CPM level remained stable after freeze drying and correlated well with the intensity of the bands observed on the autoradiograph shown in Fig. 2.
Based on autoradiographs and CPM, the reconstituted lyophilized "TcII" sample appears virtually identical to the native liquid preparation. Any quantitative differences in CPM reflect normal sample variations seen in RIPA. The ”TcI” bulk material was tested in RIPA either in its native liquid form or as a reconstituted FD sample (Fig. 2). Based on autoradiographs and CPM, the reconstituted lyophilized "TcI" sample appeared virtually identical to the native sample (as was the case for the
”TcII” material) and quantitative differences in CPM reflect normal variation seen in RIPA. Taken together, these results supported a decision to formulate the proposed candidate materials as freeze dried preparations.
Preparation of the proposed reference standard 09/186. Approximately 1.4 L of the defibrinated plasma pool representing the T. cruzi II region were sent to the NIBSC for filling and freeze-drying.
The pool was thawed and dispensed in 0.5 mL aliquots into glass ampoules, coded 09/186, and a total of 2181 ampoules were produced.
The mean fill weight for 93 ampoules was 0.5152 g (coefficient of variation (CV) of 0.20%). 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 0.04% (CV of 13.87%). Sixty eight ampoules were rejected during the production process, 40 ampoules were held for accelerated degradation studies and 1962 ampoules were stored at -20oC.
Preparation of the proposed reference standard 09/188. Approximately 0.8 L of defibrinated plasma pool representing the T. cruzi I region were sent to NIBSC for filling and freeze-drying. The pool was thawed and dispensed in 0.5 mL aliquots into glass ampoules coded 09/188, and 1307 ampoules were produced.
The mean fill weight for 52 ampoules was 0.5156 g (CV of 0.41%). 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 0.09% (CV of 8.28%). Thirty three ampoules were rejected during the production process, 40 ampoules were held for accelerated degradation studies and 1168 ampoules were stored at -20oC.
Description of study samples. Each participating laboratory received two sets of samples comprising seven coded ampoules (A, B, C, D, E, F and H). Sample G was retracted during the study. A brief description of each sample, the study codes, batch codes and the reactivity in various immunoassays prior to freeze-drying are given in Table 1. Duplicates of 09/186 (A and E) and 01/576 (B and F) were included in the sets to provide an independent measure of within-laboratory variability. All samples tested negative for antibodies to HIV 1/2 and Hepatitis C, and Hepatitis B surface antigen. Ethical approval was obtained from the Human Materials Advisory Committee at NIBSC approving the processing of the candidate reference standards (09-002SR). Finally, all study samples were distributed as FD preparations on dry ice by courier to the participants of the CS. Additional samples of 09/186 and 09/188 were used to analyse the stability of the preparations by two participant laboratories.
Serodiagnostic tests. The tests used by the participants are summarized in Table 4. They were assigned a code in roman numbers, which is used to present results without disclosing their identity in the study. The participants were asked to analyse the samples with tests they currently use for blood screening, investigation or serodiagnosis. A total of 42 different tests were used. Thirty tests are commercially available: EIA (n=16), IFA (n=4), IHA (n=4), RIC (n=4), one PAG and one ChLIA.
Some tests were marketed under different trade names in different countries in which case they were grouped together for the analysis of the results. Eleven in-house tests were used: EIA (n=2), IFA (n=5), western blots (n=3) and one RIPA. The RIPA, which has a qualitative and a quantitative aspect, was used only used in the pilot study to confirm results of screening tests such as IFA, HAI, PAG and EIA.
Two types of methods were distinguished: titration methods (IFA, IHA and PAG) and EIAs including ChLIA. Titration methods yield an endpoint titre and participating laboratories that use these were requested to perform four independent runs for each method. Since EIAs and ChLIA produce a numerical response such as an absorbance or a fluorescence value, participating laboratories were requested to report the results of six sequential doubling dilutions for each sample. The antigenic composition for recombinant EIAs, ChLIA, native EIAs, IHA, PAG and IFA tests included in the study is given in Table 5.
Study Protocol. The protocol distributed to the study participants is attached in Appendix 4. Data sheets were provided to ensure that all required information was recorded. Instructions for the reconstitution and testing of samples were also provided. Participants were informed that the EIAs would be the primary tests for the calibration of the candidate standards and were requested to prepare six sequential dilutions (2x, 4x, 8x, 16x, 32x and 64x) of the reconstituted samples with the diluent
provided by each test manufacturer. The participants were requested to test the samples on at least two different days. Two fresh sets of dilutions from the same sample were run on each of the two days so that four data points were generated for each sample and respective sequential dilutions.
IFA, IHA and PAG methods were performed following the manufacturer’s sample dilution procedure.
Each sample was tested four times on at least two separate days (2 assays on day 1 and two assays on day 2). The participating laboratories reported IHAs and IFAs titres as the reciprocal of the highest sample doubling dilutions that were positive, performed according to the test kit manufacturer’s procedure.
Participating laboratories that used commercially available tests followed procedures described by the manufacturer while laboratories using in-house tests followed standard procedures.
Participating laboratories and assay codification. Twenty-four participants from 16 countries, representing national reference laboratories (n=10), research laboratories (n=5), blood establishments laboratories (n=5), diagnostic laboratories (n=3) and one national regulatory authority took part in the collaborative study. The participating laboratories were from the following countries: Argentina (n=2), Bolivia, Brazil (n=5), Chile, Costa Rica, Ecuador, France, Honduras, Japan, México (n=2), Panamá, Paraguay, Spain, Switzerland, United Kingdom and United States of America (n=3). A full list of participants and their affiliation is given in Appendix 2. One laboratory did not participate in the CS but performed the supplemental RIPA test in the pilot study.
The participants returned raw data to NIBSC for analysis together with a description of the methods used and the manufacturer’s instructions. Throughout the study, participating laboratories were identified by a randomly assigned code number to maintain confidentiality, followed by a letter representing the number of the assay performed (e.g. 10c means laboratory 10; 3rd assay). The same letter does not correspond to the same assay among the laboratories (e.g. 10c and 9c mean assay 3 for both laboratories but would not necessarily be the same test).
Data analysis. For the EIA methods, endpoint titre estimates were calculated by linear regression using the log10-transformed optical densities and cut-off values supplied by the participants. These have also been expressed in mIU mL-1 taking the potency of each candidate standard to be 1 IU mL-1. The endpoint titre is defined as the reciprocal of the highest dilution of a sample that gives a reading above the cut-off.
The relative potencies of coded duplicates of 09/186 were calculated by parallel line bioassay analysis comparing transformed assay response, optical density, to log10 concentration (37). Deviations from the fitted model (non-linearity and non-parallelism) were considered significant at the 1% level and these assays were considered as invalid. For the titration methods (IFA, IHA and PAG), the relative endpoint titres were used to express the relative potency.
All mean estimates shown in this report are unweighted geometric mean (GM) estimates. Variability between laboratories has been expressed using geometric coefficients of variation (GCV = {10s- 1}×100% where s is the standard deviation of the log10-transformed estimates). Comparisons between candidate samples were made by performing a paired t-test on log10-transformed laboratory geometric means. Comparisons between assay methods were made by performing analysis of variance on log10- transformed laboratory geometric means using Bonferroni’s multiple comparisons correction.
RESULTS
EIA and ChLIA results.
The commercial EIAs included in this study represent the commercially available anti-T.cruzi assays most frequently used by the diagnostic reference laboratories and blood establishments for detection of T cruzi antibodies. The results for these tests are summarized in Table 6, depicted in figs 3a and 3b and in Tables A1-A3 (Appendix 1). Laboratory code 10c was excluded from summary calculations due to inconsistencies with coded duplicates in some assay runs. Laboratory code 23 was excluded from summary calculations as an outlier.
Geometric mean endpoint titres ranged from 2 in laboratory 9 (code 9c) to 37 in laboratory 2 (code 2d) for 09/186, and from 3 in laboratory 9 (code 9a) to 59 in laboratory 2 (code 2d) for 09/188. For 09/186, the overall GM was calculated as 11 (GCV 104.7%; n=29) for assays using native antigen and 9 (GCV 89.4%; n=25) for assays using recombinant antigen. For 09/188, the overall GM was calculated as 17 (GCV 101.3%; n=29) for assays using native antigen and 16 (GCV 125.1%; n=25) for assays using recombinant antigen. No statistically significant differences were detected for either sample when comparing assays using native antigen to assays using recombinant antigen (p=0.077 for 09/186 and p=0.750 for 09/188). The laboratory geometric means had a statistically significant difference when comparing sample 09/186 to 09/188 (p<0.001).
Variability within assay runs for each test code was assessed using the relative potencies of coded duplicates of 09/186 (Table A3 Appendix 1), which ranged from 0.39 to 1.67 for assays using native antigen and 0.65 to 2.19 for assays using recombinant antigens. Besides some assays in laboratories 4a (Assay 4: 2.19) and 18a (Assay 1: 0.40, Assay 3: 0.39) the relative potency of the coded duplicates using either native or recombinant antigens was within the range of 0.50 – 2.00 i.e. no more than two- fold different from their expected value of 1. This is within the range of detection for titration methods where two-fold dilution steps are used. Furthermore, 95.6 % of assays showed no more than a two- fold difference in endpoint titration value between the coded duplicates (Table A1), as a measure of within-laboratory variability.
Tests IV and V showed ≥ 3 fold increase in antibody titre for 09/188 relative to 09/186 (see Table 6). The assays' antigen composition may contribute to this difference in performance compared to the other recombinant assays. They use a unique set of antigens: FP3, FP6, FP10, and TcF, and E63 respectively. Table 5 present the antigen composition of each test included in the CS.
The results for the negative samples B, D, F and H were negative in all the participant laboratories with the exception of sample D in laboratory 9, code 9f, which showed reactivity in two out of four of the method's runs. Run 1 recorded a positive result at the neat dilution, giving an endpoint titre of 2 (568.5 mIU mL-1). Run 3 recorded positive result at the 1:21:2 dilution, giving an endpoint titre of 3 (317.3 mIU mL-1). This method did not however appear to be more sensitive than others for the positive samples. No other abnormal results were observed for this laboratory and only laboratory 9 used this assay method.
Fig. 4 shows the overall information obtained in this study for all the methods used. Reactivity data for EIA tests, collected in the preparation of the candidate standards as well as in the pilot study, were
confirmed in the collaborative study for both preparations (Table 1), with sample 09/188 slightly more reactive than sample 09/186.
IFA, IHA and PAG results.
The results for the IFA, IHA and PAG methods are summarized in Table 7 and in Table A4 of the Appendix 1. Although related to IHA by methodology, PAG results were calculated separately.
Laboratory code 21d was excluded from summary calculations due to inconsistencies with coded duplicates in all assays. Laboratory code 22e was excluded from summary calculations due to inconclusive results for 09/186 (code A).
Assay run endpoint sample dilutions ranged from 0.5 in laboratory 16 (code 16b) to 38 in laboratory 5 (code 5j) for 09/186, and from 0.5 in laboratory 16 (code 16b) to 108 in laboratory 5 (code 5j) for 09/188. For 09/186, the overall GM, based on laboratory GMs, was calculated as 1.8 (GCV 57.3%;
n=12) for IFAs, 3.9 (GCV 203.3%; n=8) for IHAs and 16.0 (GCV 100.0%; n=3) for PAG. For 09/188, the overall GM was calculated as 2.2 (GCV 55.7%; n=12) for IFAs, 6.1 (GCV 183.2%; n=8) for IHAs and 20.2 (GCV 188.3%; n=3) for PAG. The between-laboratory variability was greater for IHAs when compared to IFAs. For sample 09/186 a statistically significant difference (p<0.05) was detected when comparing PAG to both IFAs and IHAs, between which no significant difference was detected. A statistically significant difference (p<0.05) was detected when comparing EIAs to IFA and IHAs, but not to PAG. For sample 09/188 a statistically significant difference (p<0.05) was detected when comparing IFAs to both PAG and IHAs, between which no significant difference was detected. A statistically significant difference (p<0.05) was detected when comparing EIAs to IFA and IHAs, but not to PAG. The laboratory geometric means had a statistically significant difference (p<0.05) when comparing sample 09/186 to 09/188.
Variability within assays was quantified using the relative endpoint titres of coded duplicates of 09/186 (Table A4, Appendix 1), which ranged from 0.50 to 2.00 for IFAs, 0.50 to 1.84 for IHAs and 1.00 to 1.19 for PAG.
Although the IFA, IHA and PAG titration methods were not used as part of the calibration of the candidate standards, they were included for confirmation of the reactivity of the samples in these tests.
The negative samples (coded ampoules B, D, F and H) were all reported as not reactive.
Western blot results.
The results are given in Table 8 and Figs 5 and 6. As part of the collaborative study, participants 16 and 20 submitted results from TESA blot and participant 7 submitted results from an in-house western blot. Prior to blotting, samples were diluted 1:100 (participants 7 and 16) and 1:200 (participant 20).
All three participants reported a positive result for sample A, C and E and other samples were reported as negative. The criteria for a positive result varied between participants. Data from these tests could not be analysed for variance. In the chronic stage of Chagas disease, antibodies always react with antigens of a MW of 150-160 kDa and this is considered a positive result for the TESA blot, reactivity with the 95 kDa antigen is seen but it is less common. Reactivity with bands of a MW of 32 and 45 kDa was considered positive in an in-house immunoblot to confirm Chagas disease. Thus 09/186 (A, E) and 09/188 (C) performed consistently in different immunoblot assays and there is good agreement
between the results of the participants generated by this technique. The negative samples (coded ampoules B, D, F and H) were all reported as not reactive.
Rapid immunochromatographic assay results.
Four participants (2, 9, 14 and 15) carried out RICs, representing lateral flow assays and dot blot assays and results are given in Table 9. The results of the assays carried out in the four laboratories are in good agreement. Only samples A, C and E were detected as positive. The four different RIC formats used by the participants laboratories gave comparable outcomes for samples 09/186 and 09/188. The negative samples (coded ampoules B, D, F and H) were all reported as not reactive.
Stability studies.
An accelerated degradation study of the preparations 09/186 and 09/188 was carried out after 9 months’ and 21 months’ storage at temperatures of -20°C, +4°C, +20°C and +37°C. Predicted losses of activity were calculated using only the results from the two assay methods that were used at both time points. Potency estimates relative to -20°C from these methods are presented in Tables 10 and 11.
For 09/186 when stored at -20°C the data show a predicted loss of 0.14% per year with the Chagatest ELISA Recombinant 3.0 test, and a predicted loss of 0.45% per year with the ELISA cruzi test. For 09/188 when stored at -20°C the data show a predicted loss of 0.45% per year with the Chagatest ELISA Recombinant 3.0 test, but no prediction of stability was possible with the ELISA cruzi test as there was no consistent pattern of loss of potency across the temperatures. Stability of both samples could not be predicted using this dataset, but the loss in potency puts both samples in a range observed for other plasma or serum derived antibody standards.
DISCUSSION
This study is the first exercise on the standardization of biological measurements applied to the detection of T cruzi antibodies involving laboratories on a global scale. The commercial tests included in this study represent technologies currently available for diagnosis of anti-T.cruzi antibodies. These include serodiagnostic assays that are used frequently by diagnostic and blood screening laboratories.
The suitability of the two candidate reference standards (preparations 09/186 and 09/188) for determination of the analytical sensitivity of the various anti-T. cruzi test formats was assessed in the study. Reactivity data collected in the selection of the candidate standards as well as in the pilot study were confirmed in the collaborative study for both preparations, with sample 09/188 shown to be slightly more reactive than sample 09/186. Considerable differences in the tests efficiency were detected.
The candidate standards were selected to be representative of endemic regions where T. cruzi I (09/188) and T. cruzi II (09/186) are predominant (see selection of blood donor plasma samples, page 5). Human blood plasma units used to prepare these materials were donated by individuals from those geographical regions respectively. However, although efforts were made to isolate the parasites from the blood of the individual donors, confirmation of the T. cruzi genotype remains unsuccessful to date. Thus, although plasma samples were collected from endemic regions with high prevalence of TcI (09/188) or TcII (09/186), it cannot be confirmed that the preparations contain only the lineage TcI or the lineage TcII. This
difficulty had already been considered during discussions at the WHO Consultation in 2009 since parasitemia in the chronic phase of the disease is usually very low or absent.
It is therefore recommended that the proposed reference standards are used concomitantly. At least a similar range of sequential duplicate dilutions of 09/186 and 09/188 as in this study (1:2 to 1:64) is also encouraged for the evaluation of the analytical sensitivity of new tests. The protocol adopted for the WHO collaborative study is attached to this Report. In general, it is assumed that the higher the endpoint titre (or dilution of the standard) that gives a positive result, the higher the analytical sensitivity of the assay under test.
It is therefore expected that the use of the reference standards proposed will facilitate the control of analytical sensitivity of commercial and in-house tests globally and will help to improve the quality of serological methods developed for the diagnosis and screening of Chagas disease. The comparative data generated in the study may allow an initial evaluation of the current “state of the art” sensitivity of assays but further information and investigation is required.
RECOMMENDATIONS
The reactivity in EIA was used as a guide to establish the unitage mL-1 for the calibration of the candidate standards in the CS. Based on the data obtained, presented in this Report, it is proposed that preparations 09/186 and 09/188 are established by the Expert Committee on Biological Standardization as the 1st WHO International Reference Standards (IS) for anti-T. cruzi antibodies, as follows:Preparation 09/186, defined as the anti-T. cruzi antibody Standard representative of the region where T. cruzi II is predominant, with an assigned arbitrary unitage of 1 ("TcII") International Unit mL-1 for the undiluted concentration after reconstitution.
Preparation 09/188, defined as the anti-T. cruzi antibody Standard representative of the region where T cruzi I is predominant, with an assigned arbitrary unitage of 1 ("TcI") International Unit mL-1 for the undiluted concentration after reconstitution.
These standards, containing 0.5 mL freeze dried plasma, will be distributed together, with an assigned unitage of 0.5 IU per ampoule. The unitage to be associated with the endpoint titre can thus be taken as the reciprocal value of the titre, and this will inform the user of the relative strength of 09/186 and 09/188 in the assay of choice.
Information on the characteristics of the proposed standards and intended use will be provided with the Instructions for Use accompanying the distribution of the samples. The report of the collaborative study containing full details on the reactivity of both preparations will be available at the WHO website (http://www.who.int/bloodproducts/catalogue/en/index.html ).
ACKNOWLEDGMENTS:
We gratefully acknowledge the important collaboration of the participating laboratories in the WHO collaborative study. Our gratitude is also expressed to the experts participating in the WHO Consultations on International Biological Reference Preparations for Chagas Diagnostic Tests for their fundamental contributions to the development of this project and to Dr Amadeo Sáez-Alquezar, Chair of the Technical Group II on Prevention of transfusion and organ transplantation transmission of T. cruzi), WHO Programme on Control of Chagas Disease, for his expert review of the Report. We would also like to thank the Standards Processing Division team at NIBSC for processing and distribution of the candidate and trial candidate preparations.
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Table 1: Description of samples used in this study and reactivity prior to freeze drying Study
Codea
NIBSC code
Sample description Reciprocal end point titreb
IFAc IHAd,e PAGf
EIAsg,h,i A, E 09/186 Freeze dried seropositive defibrinated plasma
representing the TcII predominant region in Latin America
2 2;23; 27 4; 16; 2
B, F 01/576 Normal serum from 7 donors ND ND ND
C 09/188 Freeze dried seropositive defibrinates plasma representing the TcI predominant region in Latin America
3 4; 16; 32 4; 32; 2
D 83/571 Normal serum from 1 donor ND ND ND
H 82/582 Normal serum from 1 donor ND ND ND
a: sample G was removed prior to start of the study.
b: GM taken from four test values.
c: Imunocruzi® (BioMérieux S.A.).
d: Hemacruzi® (BioMérieux S.A.).
e: Chagatest HAI (WienerLaboratorios S.A.I.C.).
f: Seródia®(Fujirebio Inc.).
g: bioelisa Chagas (BiokitS.A.).
h: Chagatek (Laboratório Lemos S.R.L.).
i: Chagatest ELISA (WienerLaboratorios S.A.I.C.).
ND: not done
Table 2: Potencies of 09/186 and 09/188 (freeze-dried) relative to 09/186 and 09/188 (liquid)
Lab code Method 09/186 09/188
Run 1 Run 2 GM Run 1 Run 2 GM
2a EIA 0.965 1.139 1.049 1.052 1.033 1.042
2i IHA 1.000 0.707 0.841 2.000 1.000 1.414
4c IFA 0.707 1.414 1.000 2.000 5.657 3.364
4d IHA 1.000 1.000 1.000 1.000 0.707 0.841
5a EIA 0.949 0.984 0.966 0.928 0.995 0.961
5c EIA 0.927 1.004 0.965 0.943 0.916 0.930
5f EIA 0.991 0.976 0.984 1.019 0.931 0.974
5h IFA 1.000 1.000 1.000 1.000 1.000 1.000
5i IHA 1.000 1.000 1.000 1.000 1.000 1.000
5j PAG 1.414 1.414 1.414 1.000 1.000 1.000
Shaded cells represent EIAs that use recombinant antigen; EIAs represented by transparent cells use native antigen. Diagonal pattern filled cells represent PAG assays.
Table 3: Reactivity of 09/186 and 09/188 in RIPA before and after freeze drying, expressed in counts per minute relative to the positive control
NIBSC code
Counts per minute in RIPA (% of positive control)
Native sample Reconstituted FD sample Positive control and negative control neat 1:10 1:100 neat 1:10 1:100
09/186 12045 (88)
7245 (53)
5086 (37)
12010 (88)
7931 (58)
5742 (42)
13723 2945 (21) 09/188 9458
(92)
6558 (64)
4788 (46)
11766 (109)
6361 (62)
3404 (33)
10323 2450 (24)
FIGURE 1
Legend to Fig. 1: Reactivity of liquid and FD samples of 09/186 and 09/188 in TESA blot.
FD reconstituted 09/186 sample in lane 1-4: diluted 1:200, 1:400, 1:800 and 1:1600. Liquid 09/186 sample in lane 5-8: diluted 1:200, 1:400, 1:800 and 1:1600; FD reconstituted 09/188 sample in lane 9- 12: diluted 1:200, 1:400, 1:800 and 1:1600. Liquid 09/188 sample in lane 13-16: diluted 1:200, 1:400, 1:800 and 1:1600; lane Ch+: positive control diluted 1:200.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ch
150 160
MW kD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ch+
FIGURE 2
A 1 2 3 4 5 6 7 8 9 10 11 12 B 1 2 3 4 5 6 7 8 9 10 11 12
Legend to Fig 2. Autoradiograph results of the RIPA with liquid and FD samples of 09/186 and 09/188. Numbers on the lefthand side indicate MW in kDa.
Fig 2A: Negative controls in lane 1-3; Liquid 09/186 sample in lane 4-6: neat and diluted 1:10 and 1:100; FD reconstituted 09/186 sample in lane 7-9; and positive controls in lane 10-12.
Fig 2B: Negative controls in lane 1-3; Liquid 09/188 sample in lane 4-6: neat and diluted 1:10 and 1:100; FD reconstituted 09/188 sample in lane 7-9; and positive controls in lane 10-12.
Table 4: Tests used in the collaborative study
EIA with native, crude or purified T. cruzi antigens (n=29) In-house
ELISA cruzi (BioMérieux Brasil S.A.)
EIE Chagas Bio-manguinhos (Fundação Osvaldo Cruz Biomanguinhos) Test ELISA para Chagas III / CERTEST Chagas ELISA Test (GrupoBios) Hemagen® Chagas EIA (Hemagen® Diagnostic Inc.)
CHAGAS TEST (Instituto de Investigaciones de la Ciencia en Salud de Paraguay) ORTHO® T. cruzi ELISA Test System (Ortho-Clinical Diagnostics Inc.)
Biozyma ELISA Chagas / Chagatek Elisa /Accutrak Chagas Microelisa Test System (Laboratório Lemos S.R.L.) Chagascreen ELISA / Chagatest ELISA (Wiener Laboratorios S.A.I.C.)
EIA with recombinant T. cruzi antigens (n=2425) bioelisa Chagas (Biokit S.A.)
Enzyme Chagas (Interbiol S.A.)
Biozyma ELISA recombinante / Chagatek ELISA recombinante / Accutrack Chagas recombinante Microelisa Test System (Laboratório Lemos S.R.L.)
Novagnost® Chagas IgG (NovaTec Immunodiagnostica GmbH) Pathozyme® Chagas (Omega Diagnostics Ltd.)
Chagas ELISA IgG+IgM (Vircell S.L.)
Chagatest ELISA recombinante V3 (Wiener Laboratorios S.A.I.C.) Chagatest ELISA recombinante V4 (Wiener Laboratorios S.A.I.C.) ChLIA with recombinant T. cruzi antigens (n=1)
PRISM CHAGAS (Abbott Diagnostics Division) IFA (n=12)
In-house
Inmunofluor® Chagas (Biocientífica S.A.) Biognost® Trypanosoma IgG IFA (Bios GmbH) Imunocruzi® (BioMérieux Brasil S.A.)
IFI Chagas Bio-manguinhos (Fundação Osvaldo Cruz Biomanguinhos) IHA (n=10)
Hemacruzi® (BioMérieux Brasil S.A.) Chagas/HAI (Interbiol S.A.)
HAI Chagas (Polychaco S.A.I.C.)
Chagatest HAI / Chagatest HAI screening A-V (Wiener Laboratorios S.A.I.C.) PAG (n=3)
Seródia®-Chagas PAG (Fujirebio Inc.) RIC with recombinant T. cruzi antigens (n=5)
Chagas Stat-Pak® Assay (Chembio Diagnostic Systems Inc.) OnSite Chagas Ab Combo Cass. (CTK Biotech Inc.) Simple Chagas/Stick Chagas (Operon S.A.)
ImmunoComb II® Chagas Ab (Orgenics Ltd.) Western blot assays (n=3)
In-house, Western blot In-house, TESA blot
(n= total number of datasets received)
Table 5 Antigen composition of quantitative antibody tests
*Transparent cells are IFAs; shaded cells are IHAs; diagonal pattern filled cells are PAG Test code XXVI represents five different in-house IFA tests
Test code EIAs and ChLIA Recombinant antigen composition I TcD, TcE, Pep2 and TcLo1.2
II TcD, TcE, Pep2, SAPA and 5º waiting patent III SAPA,Ag1, Ag2, Ag13, Ag30 and Ag36 IV TcF, FP3, FP6 and FP10
V E63
VI TcF
VII Miranda/76 clone: SAPA, Ag1, Ag2, Ag13, Ag30 and Ag36 VIII T.cruzi Y: FRA, B13 and 1F8
IX SAPA, Ag1, Ag2, Ag13, Ag30 and Ag36
Test code Native EIAs strain type
X T. cruzi Y and CL
XI Tulahuen
XII T. cruzi Y and Tulahuen XIII Tulahuen and Mn
XIV Tulahuen
XV T. cruzi Y
XVI T. cruzi Y
XVII Tulahuen
XVIII T. cruzi Y
XIX Tulahuen+Mc+Dm28
Test code IFA, IHA and PAG assays strain type)*
XX T. cruzi Y and Tulahuen
XXI Tulahuen
XXII T.cruzi RF XXIII T. cruzi Y
XXIV Soluble extract native antigen Mexico (Zacatecas, Oaxaca y Yucatan)
XXV Tulahuen
XXVI T. cruzi Y, Jenifer, Tulahuen, Tulahuen+Mc+Dm28, T. cruzi Y XXVII T. cruzi Y
XXVIII T. cruzi RA (Argentina) XXIX T. cruzi ATCC 30013
XXX T. cruzi Y
Table 6: Geometric mean endpoint titre estimates from EIAs and ChLIA.
Lab code Test code
09/186 09/188
GM mIU mL-1 GCV GM mIU mL-1 GCV
2a I 7 143.60 12.5% 8 125.00 ---
5a I 6 174.44 8.8% 5 202.05 18.1%
9a I 2 408.25 24.2% 3 368.89 22.5%
13a I 5 206.71 14.2% 5 204.12 26.4%
14 I 5 202.05 18.1% 5 204.12 ---
Mean I 5 211.89 5 207.88
2e II 22 46.20 12.1% 37 27.28 ---
3a II 8 121.37 6.1% 27 36.37 ---
5b II 11 90.84 16.4% 27 36.53 20.4%
Mean II 13 79.87 30 33.09
3b III 7 138.88 14.7% 13 74.54 ---
4a III 11 90.99 43.9% 32 30.81 47.0%
5d III 20 48.85 6.0% 45 22.03 8.4%
6a III 13 78.91 49.1% 24 42.51 32.9%
7b III 7 133.48 36.1% 15 67.42 ---
11a III 20 50.38 54.5% 20 48.91 8.8%
12 III 11 91.11 51.1% 27 37.16 27.4%
15 III 10 96.52 51.0% 19 52.63 ---
16a III 8 129.17 31.6% 16 61.09 37.7%
17b III 17 58.16 22.5% 29 34.40 13.7%
21c III 7 142.43 34.5% 14 73.13 ---
Mean III 11 89.85 22 46.37
1a IV >15 >65.53 >88.7% >54 >18.35 >11.6%
2c V 5 188.98 38.1% 15 66.23 ---
9f VI 7 137.33 58.1% 10 97.82 31.7%
19 VII 5 201.02 11.5% 11 91.68 16.2%
9c VIII 2 500.00 0.0% 5 200.00 0.0%
3c IX 24 40.86 18.9% 44 22.78 ---
