HIV SIMPLE/RAPID ASSAYS: OPERATIONAL CHARACTERISTICS

Transcription

1 WHO/BCT/2.7 HIV SIMPLE/RAPID ASSAYS: OPERATIONAL CHARACTERISTICS (PHASE I) REPORT 2 WHOLE BLOOD SPECIMENS Blood Safety and Clinical Technology January 22 World Health Organization

2 GENEVA Copyright World Health Organisation, 22 This document is issued by WHO for general distribution. The document is not a formal publication of the World Health Organization (WHO), and all rights are reserved by the Organization. This document may not be reproduced, in part or in whole, without written approval from the contact person listed below. This document must not be used in conjunction with commercial or promotional purposes. Neither WHO nor the Institute of Tropical Medicine, Antwerp, Belgium warrant or represent that the evaluations conducted with the HIV test kits referred to in this document are accurate, complete and/or errorfree. WHO and the Institute of Tropical Medicine disclaim all responsibility and liability for any use made of the data contained herein. The mention of specific companies and of certain manufacturers products does not imply that they are endorsed or recommended by WHO in preference to others of a similar nature that are not mentioned. Contact: Dr. G. Vercauteren Blood Safety and Clinical Technology WHO 2, Avenue Appia 2 Geneva 27 Switzerland.

3 WHO/BCT/2.7 HIV SIMPLE/RAPID ASSAYS: OPERATIONAL CHARACTERISTICS (PHASE I) Table of Contents WHOLE BLOOD SPECIMENS. Introduction 2. Background information. Laboratory aspects of HIV testing. A brief overview.2 Quality assurance. Safety 4. Materials and Methods 4 4. Assays (test kits) evaluated Evaluation panel 7 4. Laboratory testing Data analysis Sensitivity, specificity, confidence limits and predictive values of HIV tests Interreader variability 4.4. Sensitivity in seroconversion panels Additional analyses. Assay evaluations 9 Table : General characteristics and operational aspects Table 2: Comparison of whole blood antihiv assays with reference tests 2 Table : Detailed operational aspects Table 4a: Technician s appraisal of the test kits Table 4b: Calculation of ease of performance 6 Table : Suitability for use in small laboratories 7 Table 6: Performance on seroconversion panels 8 Figure : Relative performance on seroconversion panels 9 Explanatory notes for Tables 6 and Figure 2 6. Annexes 2 Annex : Algorithm for characterization of the WHO HIV panel 2 Annex 2: List of assay manufacturers /distributors addresses Additional reading Acknowledgements Note 24

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5 WHO/BCT/2.7 Page INTRODUCTION Since 988, the World Health Organization (WHO), conscious of the need to advise Member States on the laboratory diagnosis of HIV, has been providing member states with objective assessments of the operational characteristics of commercially available HIV tests. This continuing program is carried out by the WHO Collaborating Centre at the Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium and is coordinated by the Department of Blood Safety and Clinical Technology, WHO, Geneva in conjunction with UNAIDS. The WHO evaluation scheme provides information that is useful for decisionmakers to allow them to make the most suitable choices. The assessment focuses on the operational characteristics of these assays, such as sensitivity, specificity, and positive and negative predictive values. Additional information related to incubation temperatures, shelflife and ease of use are also provided. Report 2, dealing with the evaluation of the major operational characteristics of commercially available assays to detect antibodies to HIV in whole blood specimens, presents assessments of the following seven assays carried out between May 2 and September 2: Determine HIV/2 (Abbott Laboratories) InstantScreen Rapid HIV/2 Assay (GAIFAR GmbH) ADVANCED QUALITY Rapid HIV Test (InTec Products Inc.) MedMira Rapid HIV Test (MedMira Laboratories Inc.) First Response HIV/HIV2 WB (PMC Medical Pty. Ltd) CAPILLUS HIV/HIV2 (Trinity Biotech PLC) UniGold HIV (Trinity Biotech PLC) This report provides data of phase I of the WHO coordinated evaluation of seven antihiv simple/rapid (S/R) test kits using whole blood specimens. Phase II, is planned for 22, and will include field testing. Copies of this report are available on request from the Blood Safety Team, Department of Blood Safety and Clinical Technology, World Health Organization, 2 Geneva 27, Switzerland. The report is also available on the BCT section of the WHO website 2. BACKGROUND In the last few years there have been a number of improvements of S/R tests for the detection of HIV antibodies (antihiv). One important advancement has been the development of assays that detect HIV antibodies in human whole blood, whereas previously samples of serum or plasma were required. Further refinements have meant that the sensitivity and specificity of S/R tests has increased. These developments may be of particular use in voluntary councelling and testing (VCT) and/or prevention of mothertochild transmission (PMTCT) initiatives, as it is recognised that an individual s knowledge of their HIV status is the first step in halting the spread of the disease and for seeking appropriate clinical care. For such initiatives to be effective, whether carried out in central

6 WHO/BCT/2.7 Page 2 laboratories or small health centres, it is necessary to identify the most appropriate assay for their particular circumstances. The need for reliable tests that can be used in situations with limited laboratory facilities continues to drive the development of S/R tests for antihiv in recent years. Transport to rural areas is less of a logistical problem when only the S/R tests themselves are required and there is no necessity for the delivery of numerous bulky items of equipment, as would be required for other test formats, such as ELISAs. These tests are simple in that they need little or no equipment, have few manipulation steps, can be read visually and are suitable for use on single or low numbers of specimens. Such tests can be carried out by less experienced staff, nonetheless the personnel should have a minimum of training with regard to the operation and interpretation of results, have undergone safety training and operate within a quality assurance programme. The tests are also rapid in that the time required to complete the assay is short, frequently less than minutes. The development of S/R tests that can detect HIV antibodies in whole blood in addition to serum and plasma has allowed the use of these assays in situations where the necessity for equipment such as a centrifuge is impracticable, eg rural health clinics or voluntary councelling and testing services (VCT), thus even the need for electricity is obviated. In such settings it is possible to carry out the testing immediately after the blood specimen is taken, thus reducing further the waiting time for results. The possibility of error in specimen labelling is also reduced as the original specimen is used and there is no necessity to remove an aliquot of the serum into a new vial. Nonetheless, clerical error may still occur if the system is not well organised and a quality system should be in place. A disadvantage of whole blood specimens is that they deteriorate on storage and therefore cannot be used for further testing at a later date, in which case a serum specimen would be required. Whether the whole blood sample is a venous sample or obtained by fingerprick, it is essential that the site from which the sample is taken should be covered to reduce risk of infection and that proper waste disposal facilities for sharps are available. The majority of S/R tests can be carried out at ambient temperature and often the kits may be stored at temperatures between 2 C and C, some even up to 4 C. In most cases the kits contain the reagents readyforuse which, combined with the ease of use and incorporation of an internal control, helps to reduce technical errors. The characteristics of such assays lend themselves readily for use in resourcepoor settings where the infrastructure and human resources do not necessarily support the use of ELISA techniques and also in situations where it is necessary to give the patient the result immediately. Although the cost per test of S/R tests may be higher than that for ELISAs tests, when the latter are used to test small numbers at a time the cost can become greater. Indeed, when the above factors are taken into account it can be seen that the results obtained in such settings may be more reliable when S/R tests as opposed to ELISA techniques are employed and the use of S/R tests to be the optimal choice. There are situations where S/R tests may prove more appropriate for use in what would normally be considered those more suitable for ELISAs, eg blood donor screening, especially when using S/R tests of high quality. As an example, in an emergency situation where the time required for an ELISA test would be too great, the speed of use for a rapid test may make it more favourable to an ELISA test. In the same situation, the higher comparative cost of carrying out a single ELISA test compared with a single S/R test may not be acceptable. Thus the flexibility and speed of reliable S/R tests can make them very costeffective, and possibly preferable, in many situations.

7 WHO/BCT/2.7 Page. LABORATORY ASPECTS OF HIV TESTING. A brief overview The diagnosis of HIV infection is usually made on the basis of the detection of antibodies to HIV. Serological tests for detecting antibodies to HIV are generally classified as screening tests (sometimes referred to as initial tests) or confirmatory tests (sometimes referred to as supplemental tests). Initial tests provide the presumptive identification of antibodypositive specimens, and supplemental tests are used to confirm whether specimens found reactive with a particular screening test contain antibodies specific to HIV. When a single screening assay is used for testing in a population with a very low prevalence of HIV infection, the probability that a person is infected when a positive test result is obtained (i.e., the positive predictive value) is very low, since the majority of people with positive results are not infected. This problem occurs even when a test with high specificity is used. Accuracy can be improved if a second supplemental test is used to retest all those samples found positive by the first test. Those found negative by the test are considered negative for antibodies to HIV. Confirmation of the results obtained with alternative specimens may pose some challenges. For serum/plasma specimens the most commonly used confirmatory test was the Western blot (WB), however its use has proven to be very expensive and can, under some conditions, produce a relatively large number of indeterminate results. Similar assays, generically called line immunoassays (LIAs), based on recombinant and/or synthetic peptides capable of detecting antibodies to specific HIV and/or HIV 2 proteins, have been developed, eg INNOLIA and PeptiLAV. In general these assays produce fewer indeterminate results as compared with WB but are equally expensive. Studies have shown that combinations of ELISAs or S/R assays can provide results as reliable as the WB at a much lower cost. WHO and UNAIDS therefore recommend that countries consider testing strategies that use ELISAs and S/R assays rather than ELISA/WB for HIV antibody detection..2 Quality assurance All laboratories carrying out HIV tests, should have a wellfunctioning quality assurance programme. It is most important that quality assurance procedures be stringently applied so as to maximize the accuracy of the laboratory results. Procedures for detecting both (technical) laboratory and clerical errors must be included in all protocols, for example, procedures that guarantee that the correct results are communicated to the individuals seeking to know their HIV status. External Quality Assessment Schemes (EQAS) are available for HIV serology and it is recommended that laboratories participate in an EQAS, wherever possible, at least once a year to monitor their performance.. Safety The testing of whole blood specimens should be performed in such a manner as to minimize occupational risk, in the same way as that for serum and plasma specimens. Guidelines for good laboratory practice have been developed that, if followed, will ensure safety and keep laboratory accidents to a minimum. For further details see the Laboratory Biosafety Manual, second edition, World Health Organization, Geneva, 99 (ISBN ) and the Communicable Diseases

8 WHO/BCT/2.7 Page 4 Surveillance and Response section of the WHO website, where information on laboratory biosafety and transport of infectious substances may be found. 4 Materials and Methods 4. Assays (test kits) evaluated Test kits for these assessments were kindly provided to WHO free of charge by each of the manufacturers of the assays under evaluation. The manufacturers were invited to visit the site at which the assessments were to be conducted in order to provide any required training and to ensure that the assays were performed correctly by the laboratory staff carrying out the evaluation of their assays. All of the test kits can be used with serum serum/plasma specimens, however in this evaluation the tests were carried out on a WHO panel of whole blood specimens and on eight commercial serum seroconversion panels (see section.2). Determine HIV/2 (Abbott Laboratories, Il, USA) Product code 7D2 An invitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. For testing whole blood specimens, an extra step is required, ie to add chase buffer after the addition of the blood sample. InstantScreen Rapid HIV/2 Assay (GAIFAR GmbH, Potsdam, Germany) Product code not available An invitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. The testing procedure is identical for all three specimen types. Two versions of the assay were tested in the evaluation, InstantScreen Rapid HIV/2 assay Generation and InstantScreen Rapid HIV/2 assay Generation 2. The assays differ in formulation, binding procedure and concentrations of the antigen components of the assay. The Generation assay has been replaced by the Generation 2 assay. (Image of the test device is not available)

9 WHO/BCT/2.7 Page ADVANCED QUALITY Rapid HIV Test (InTec Products Inc., Xiamen, China) Product code ITP22TC4 An enhanced rapid immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. For serum and plasma samples the sample diluent is added immediately after the specimen whereas for whole blood specimens it is necessary to wait approximately 2 seconds before addition of the sample diluent. MedMira Rapid HIV Test (MedMira Laboratories Inc. Toronto, Canada) Product code not available An invitro immunofiltration test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasm and whole blood. For serum/plasma specimens, the sample is added directly to the device membrane. For whole blood specimens the sample is added to a filter unit that is placed on the device. The filter unit is discarded after addition of sample and buffer but prior to addition of conjugate solution. Images shown are of two versions of the MedMira Rapid HIV test provided by the company. The single test dot format, version, was tested in the WHO whole blood panel. The 2line format, version 2, was a later version of the assay which was used for testing with the commercial seroconversion panels. Version Version 2

10 WHO/BCT/2.7 Page 6 First Response HIV/HIV2 WB (PMC Medical Pty. Ltd., Daman, India) Product code DD/8 An invitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. 2µl of serum/plasma or µl whole blood is required for the assay, otherwise the testing procedure is the same for all specimen types. CAPILLUS HIV/HIV2 (Trinity Biotech PLC., Bray, Ireland) Product code 648G A latex agglutination test for the detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. The testing procedure is the same for all three specimen types. At the time of the evaluation the manufacturer stated that the kit was validated for whole blood specimens although this was not stated in the kit insert. The kit insert has since been updated detailing the testing procedure for serum, plasma and whole blood.

11 WHO/BCT/2.7 Page 7 UniGold HIV (Trinity Biotech PLC., Bray, Ireland) Product code 262 An invitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV and HIV 2 in human serum, plasma and whole blood. The testing procedure is the same for all three specimen types. Two versions of the UniGold HIV test, one containing peptide antigen and one containing recombinant antigen, were available at the time of the evaluation. The version included in this evaluation is that which contained recombinant antigen. The UniGold HIV peptide test is no longer in production. 4.2 Evaluation panel Two hundred and fifty (2) whole blood samples were collected into EDTA tubes along with a matched serum sample from individuals who gave consent to be included in this trial at ITM, Antwerp, Belgium. Eighty antihiv positive and 7 antihiv negative samples were prospectively collected for this evaluation. The composition of the panel collected is shown below. Composition of WHO HIV whole blood and matched serum panel, Phase AntiHIV positive AntiHIV Negative Total % 68% % For characterization, the matched serum samples were screened with 2 reference ELISAs, Vironostika HIV UniForm II plus O (Organon Teknika) and the Enzygnost AntiHIV /2 Plus (Behringwerke AG). Specimens nonreactive in both assays were considered antihiv negative. Specimens that

12 WHO/BCT/2.7 Page 8 were initially reactive in either assay were repeated in duplicate. Specimens repeatedly reactive in either or both ELISAs were further characterised using an immunoblot, InnoLIA HIV Confirmation (Innogenetics). The algorithm for the characterization of the matched serum samples is presented in Annex. In addition to the antihiv positive and negative samples, eight commercial serum/plasma seroconversion panels from Boston Biomedica Inc. (BBI) were tested in each of the 7 assays evaluated. 4. Laboratory testing The whole blood specimens included in the WHO whole blood panel were tested immediately after collection and in accordance with each manufacturer s instructions. All assay runs were performed by one operator and visual interpretations of the results were made independently by three technicians. Whole blood specimens in the WHO HIV panel that gave initial results discordant from the reference results on the matched serum specimens were retested in duplicate. The results that occurred 2 out of times were recorded as the final results. Samples from the commercial serum/plasma seroconversion panels giving discordant results were not retested. 4.4 Data analysis 4.4. Sensitivity, specificity, confidence limits (CL) and predictive values of antihiv tests The formula for calculation of sensitivity, specificity and predictive values is represented diagrammatically in the table below. Calculation of sensitivity, specificity and predictive values True antihiv status Results of assay under evaluation a Truepositives c Falsenegatives ac b Falsepositives d Truenegatives bd ab cd Sensitivity = a/(ac) Specificity = d/(bd) Positive predictive value = a/(ab) Negative predictive value = d/(cd) Sensitivity: is the ability of the assay under evaluation to identify correctly specimens that contain antibody to HIV (reference assays positive). Thus, sensitivity is the number of true positive specimens recognized by the assay under evaluation as positive, divided by the number of specimens identified by the reference assays as positive, expressed as a percentage. Specificity: is the ability of the assay under evaluation to identify correctly specimens that do not contain antibody to HIV (reference assays negative). Thus specificity is the number of true negative

13 WHO/BCT/2.7 Page 9 specimens recognized by the assay under evaluation as negative, divided by the number of specimens identified by the reference assays as negative, expressed as a percentage. NOTE: Specimens that gave indeterminate results with the assays under evaluation were included in the analyses as a false result. Confidence limits (CL): the 9% confidence limits are a means of determining whether observed differences in sensitivity or specificity between assays are significant or not. Exact 9% confidence limits for Binomial proportions were calculated from the Fdistribution (Armitage P. and Berry G. Statistical Methods in Medical Research, 2 nd Edition. Blackwell Scientific Publications, Oxford, 987, page 9). Predictive Values: The positive predictive value (PPV) is the probability that when the test is reactive, the specimen does contain antibody to HIV. This may be calculated in two ways:. using the simple formula a/(ab) which will give an approximate value (see Table C). 2. using the more precise formula which takes the prevalence of HIV in the population into account PPV= (prevalence)(sensitivity) (prevalence)(sensitivity) ( prevalence)( specificity) The negative predictive value (NPV) is the probability that when the test is negative, a specimen does not have antibody to HIV. This may be calculated using:. the simple formula d/(cd) which will give an approximate value (see Table C). 2. the more precise formula which takes the prevalence of HIV in the population into account: NPV= ( prevalence)(specificity) ( prevalence)(specificity) (prevalence)( sensitivity) The probability that a test will accurately determine the true infection status of a person being tested varies with the prevalence of HIV infection in the population from which the person comes. In general, the higher the prevalence of HIV infection in the population, the greater the probability that a person testing positive is truly infected (i.e., the greater the positive predictive value [PPV]). Thus, with increasing prevalence, the proportion of falsepositive decreases; conversely, the likelihood that a person showing negative test results is truly uninfected (i.e., the negative predictive value [NPV]), decreases as prevalence increases. Therefore, as prevalence increases, so does the proportion of samples testing falsenegative. However, this effect only becomes apparent at prevalence of 8% and above.

14 WHO/BCT/2.7 Page Interreader variability: The interreader variability was calculated as the percentage of specimens for which initial test results were differently interpreted (i.e. positive, negative or indeterminate) by the independent readers Sensitivity in seroconversion panels: The results obtained with commercial serum/plasma early seroconversion panels using the assays under evaluation were compared with those obtained using Enzygnost AntiHIV /2 Plus (Behringwerke AG), the assay arbitrarily designated the reference for determination of relative sensitivity in these panels. For each seroconversion series (panel) the first specimen in the sample sequence to become reactive with Enzygnost AntiHIV /2 Plus (Behringwerke AG) was assigned the value. Results from the assays under evaluation were compared with Enzygnost AntiHIV /2 Plus (Behringwerke AG) by determining the difference between the specimen assigned value and the relative position in the sample sequence of the first specimen which showed a reactive result with each of the assays under evaluation. For example, if an assay became reactive two specimens earlier in a series than Enzygnost AntiHIV /2 Plus (Behringwerke AG), the value assigned for that series in that assay was 2. Similarly, if an assay became reactive one specimen later than Enzygnost Anti HIV /2 Plus (Behringwerke AG), the value assigned was. If no specimens in the panel are positive then the assigned value was the total number of specimen in the panel. The assigned values over the 8 seroconversion series were averaged to determine a mean relative seroconversion sensitivity index for each assay and the 9% confidence limits were determined. The results for the seroconversion panels are presented in Table 6 and diagrammatically in figure Additional analyses The technical aspects of the assays under evaluation were assessed by the technician who performed the testing. These assessments, along with other selected assay characteristics, contributed to an overall appraisal of each assay s suitability for use in small laboratories. To enable comparison between assays, an arbitrary scoring system was used to rate specified assay characteristics.. Assay evaluations Results from the testing of the HIV whole blood and seroconversion panels are presented in Tables 2 and 6 and figure on the following pages. Of the seven assays, six obtained % sensitivity and one obtained 98.8% in the positive panel tested. Care should be taken when interpreting these results as only a small number of positive samples (n=8) was tested and evaluation on a larger panel including specimens from other geographical regions is required to confirm these findings. Four kits obtained specificity of %, and the other obtained 97.6%, 98.8% and 99.4% respectively. The results for both sensitivity and specificity should be interpreted carefully and by taking the 9% confidence limits (9%CL) into account. Many of these assays have been evaluated in serum/plasma panels previously by WHO and other groups. The Determine HIV/2 (Abbott), Capillus HIV/2 (Trinity Biotech) and UniGold HIV (Trinity Biotech) have been evaluated on serum/plasma panels previously by WHO. This is available on the WHO website However, the focus of this evaluation was specific in assessing the performance of these 7 S/R antihiv tests on whole blood samples.

15 WHO/BCT/2.7 Page ASSAY EVALUATIONS Whole Blood Specimens

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17 Table. General characteristics and operational aspects Name Determine InstantScreen HIV/2 Rapid HIV/2 Assay* Company Abbott Laboratories, Dainabot Co. Ltd., Tokyo, Japan GAIFAR GmbH, Potsdam, Germany ADVANCED QUALITY Rapid HIV Test InTec Products Inc., Xiamen, China MedMira Rapid HIV Test MedMira Laboratories Inc., Halifax, Canada First Response HIV/HIV2 WB PMC Medical Pty. Ltd., Daman, India CAPILLUS HIV/HIV2 Trinity Biotech PLC, Bray, Ireland UniGold HIV Trinity Biotech PLC, Bray, Ireland Assay type immunochromatographic immunofiltration immunochromatographic immunofiltration immunochromatographic agglutination immunochromatographic Solid phase membrane membrane membrane membrane membrane polystyrene latex beads Antigen type recombinant/synthetic epitopecombiantigens Specimen type optional Number of tests per kit (product code) Lot numbers evaluated (expiry date) Shelf life ( at C) Volume of serum needed (µl) Final dilution of serum Total time to perform the assay: h.min. ( specimen) whole blood serum/plasma (7D2) 629U2 (/4/2) 8 mths (2 ) none whole blood serum/plasma 2 (not available) Gen : 99 (2/2) Gen : ISO499 (2/2) Gen 2: ISO 7 (/2) 8 mths (2 to 4) 2 none membrane recombinant synthetic recombinant recombinant recombinant whole blood serum/plasma 4 (ITP22TC4) RD 979 (/2) 8 mths (2 ) /2 whole blood serum/plasma (not available) CTIA 9 (/2) year (2 ) none whole blood serum/plasma (DD/8) D2 (8/2) 8 mths (2 ) none whole blood serum/plasma (648G) G42 (//2) 2 mths (2 8) / whole blood serum/plasma 2 (262) G27 (/2/2) mths (2 27) none Page 2 Reading visual visual visual visual visual visual and/or by Capillus Digital Reader Price/test (US$) *Two versions of the InstantScreen assay were evaluated, Generation and Generation 2. visual

18 Table 2. Comparison of the whole blood HIV assays with reference test Name Final sensitivity % (9 CL) n=8 Determine HIV/2 InstantScreen Rapid HIV/2 Assay*. (9..) Gen : n= (89..) Gen 2: n=4. (9..) ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV 98.8 (9.2.). (9..) (9. ). (9..). (9..) Initial specificity % (9 CL) 99.4 (96.7.) Gen : n= (9..) Gen 2: n=8 99. (9..) 97. ( ) 9.2 (8.9 9.) 98.2 ( ). (97.9.). (97.9.) Final specificity % (9 CL) n= (96.7.) Gen : n= (9..) Gen 2: n=8. (97..) Indeterminate results %. Gen :. Gen 2:. Initial interreader variability %.6 Gen :. Gen 2:.7 PPV.% 2 6.% Gen :. Gen 2:. Gen : 7.8 Gen 2:.. (97.9.) 97.6 ( ) 98.8 ( ). (97.9.). (97.9.) NPV.% 6.%.. Gen :. Gen 2:. Gen : Gen 2:. 9% Confidence Limits. 2 Please note that, due to the formula used, small differences in specificity may result in apparently large differences in PPV in low prevalence populations. * 46 and 4 HIV positive specimens of the whole blood panel were tested in the Generation and Generation 2 InstantScreen assays, respectively, and 62 and 8 HIV negative specimens were tested in the Generation and Generation 2 assays, respectively

19 Table. Detailed operational aspects Name Determine HIV/2 InstantScreen Rapid HIV/2 Assay ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV Dimension (cm) of kit: wlh Storage conditions ( C) Incubation temperature ( C) room temperature none stated room temperature 2 room temperature room temperature room temperature Reading endpoint stability. > (h.min) Stability after dilution/ Reconstitution/opening ( C) antigen controls sample diluent conjugate substrate wash buffer expiry date (2 ) expiry date (2 ) expiry date (2 ) expiry date (2 4) expiry date (2 4) expiry date (2 4) expiry date (2 4) expiry date (2 ) expiry date (2 ) expiry date (2 ) week (2 8) week (2 8) expiry date (2 8) expiry date (2 ) expiry date (2 ) expiry date (2 8) expiry date (2 8) expiry date (2 27) expiry date (2 27) Number of sera per run Minimum maximum Number of controls per test run negative cutoff/weak positive positive blank internal control: reagent control : sample addition control * * Version of the MedMira HIV Rapid test does not contain a reagent control. The later version 2 of the assay contains a reagent control line.

20 Table. (continued) Detailed operational aspects Name Determine HIV/2 InstantScreen Rapid HIV/2 Assay Estimated time to perform one run: h. min Equipment needed but not provided in the kit : washer incubator (waterbath) spectrophotometric reader refrigerator (storage) agitator, rocker aspiration device automatic pipette ( O multichannel pipette ( O disposable tips dilution tubes/rack, microtiterplate distilled or deionised water plate covers graduated pipette; cylinder (ml) sulfuric acid/sodium hydroxide absorbent paper disinfectant gloves reagent trough timer Definition of positive results Definition of grey zone or Indeterminate result ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV / / Appearance of red control and patient bars Appearance of blue test and control dots Appearance of test and control lines Appearance of distinct red dot Appearance of pinkpurple test and control lines Appearance of any agglutination Appearance of test and control lines Not applicable Not applicable Not applicable Not applicable Not applicable * Not applicable : not provided in the kit but necessary to perform the test: : provided in the kit or not necessary to perform the test; /: use is optional. * When using the optional photometer a Threshold reading is displayed.

21 Table 4a. Technican s appraisal of the test kit Name Score Determine HIV/2 InstantScreen Rapid HIV/2 Assay ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV Number of steps in the test procedure: 2 steps steps > steps Clarity of kit instructions: good needs improvement Kit and reagent packaging and labelling: good needs improvement Total (out of possible ) Comments on the test kit Control samples should be used with this test. Sample volumes, time periods and storage conditions information in the kit insert could lead to misunderstanding. These should be defined precisely. Migration of sample/conjugate mixture not completed in all cases within time limit of 2 minutes. Red cells observed after removal of prefilter for samples Control samples should be used with this test. Outer packaging and components should be labelled with kit name and expiry date. The kit insert provided did not give details of procedure for testing whole blood specimens, however it has since been updated. Control samples should be used with this test.

22 Table 4b. Calculation of ease of performance Name Need to prepare: antigen substrate wash solution conjugate predilution of serum Stability after dilution/opening: (expiry date = ; less = ) antigen controls sample diluent conjugate substrate wash buffer Sufficient reagents wash buffer (yes = ; no = ) Item needed but not provided in the kit: reagent trough automatic/multichannel pipette dilution tubes, rack/microtiter plate distilled or deionised water plate covers graduated pipette, cylinder sulfuric acid/sodium hydroxide Technician s appraisal of the test kit (rating out of ) Determine HIV/2 InstantScreen Rapid HIV/2 Assay (Gen2) 2 ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV Total (out of possible ) Ease of performance: less easy <2 easy 2 # x # 2 very easy >2 very easy very easy very easy easy very easy very easy very easy : positive rating: reagent needs no preparation; item provided in the kit 2 : negative rating: reagenàpt needs preparation; item not provided in the kit : see table 4a

23 Table. Suitability for use in small laboratories Name Score Determine HIV/2 InstantScreen Rapid HIV/2 Assay ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV/HIV2 UniGold HIV Page 8 Sensitivity % 98 % <98% Specificity >98% 9 98% <9% Incubation temperature room t other than room t Shelflife > year $6 months # year < 6 months Storage at ambient t possible (opened kit) ambient t possible (unopened kit) 28 C required Price per test (US$) #. #2. > 2. Ease of performance very easy easy less easy Rapidity of performance: serum < min min > min Washer/agitator not needed needed Reading visual: interreader variability #% : interreader variability >% reading equipment Total (out of possible 4) Suitability for use in small laboratories: less suitable < 2 suitable # x # very suitable > 2 very suitable very suitable very suitable very suitable very suitable very suitable very suitable 2 2

24 Table 6. Performance on commercial serum/plasma seroconversion panels Panel ID Bleed date Days since first bleed Abbott HIVAG ratio* Enzygnost AntiHIV /2 Plus SR SR2 SR SR4 SR SR6 SR7 INNOLIA HIV Confirmation sgp2 gp4 p p24 p7 sgp gp6 Result OD/CO PRB neg neg neg neg neg neg neg neg neg neg neg ind neg neg neg neg >6.7 pos pos pos pos pos pos pos HIV >6.7 pos pos pos pos pos pos pos HIV >6.7 pos pos pos pos pos pos pos HIV >6.7 pos pos pos pos pos pos pos HIV >6.7 pos pos pos pos pos pos pos HIV PRB neg neg neg pos pos neg neg neg >6.7 pos pos pos pos pos pos pos 2 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV PRB >6.7 pos pos neg pos pos pos pos 2 / HIV >6.7 pos pos neg pos pos pos pos 2 HIV >6.7 pos pos neg pos pos pos pos 2 2 HIV >6.7 pos pos neg pos pos pos pos HIV >6.7 pos pos neg pos pos pos pos HIV PRB neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos neg neg neg neg >6.7 pos pos neg pos pos pos pos 2 / HIV >6.7 pos pos pos pos pos pos pos 2 HIV PRB neg neg neg neg neg neg neg neg >22,7 2.2 neg neg neg neg neg neg neg neg >6.7 pos pos neg pos pos pos pos 2 ind >6.7 pos pos pos pos pos pos pos 2 HIV >6.7 pos pos pos pos pos pos pos HIV PRB neg neg neg neg neg neg neg neg >22,7 4.8 pos pos neg pos pos neg pos neg >6.7 pos pos pos pos pos pos pos 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV >6.7 pos pos pos pos pos pos pos HIV PRB neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos neg neg neg pos neg pos ind >6.7 pos pos neg pos pos pos pos 2 2 HIV PRB neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos pos neg pos pos pos pos 2 HIV >6.7 pos pos pos pos pos pos pos 2 2 HIV Notes: *Data provided by BBI. OD/CO = optical density value of speciemn/calculated cut off value SR: Determine HIV/2 (Abbott Laboratories) SR4: MedMira Rapid HIV Test (MedMira Laboratories Inc.) SR7: UniGold HIV (Trinity Biotech PLC) SR2: InstantScreen Rapid HIV/2 Assay (GAIFAR GmbH) SR: ADVANCED QUALITY Rapid HIV Test (InTec Products Inc.) SR: First Response HIV/HIV2 WB (PMC Medical Pty. Ltd) SR6: CAPILLUS HIV/HIV2 (Trinity Biotech PLC)

25 FIGURE : RELATIVE PERFORMANCE OF ANTIHIV ASSAYS IN COMMERCIAL SERUM/PLASMA SEROCONVERSION PANELS AS COMPARED TO THE REFERENCE ASSAY (ENZYGNOST ANTIHIV /2 PLUS) Page 2. Detection Before Reference Assay 2... Determine HIV/2 InstantScreen Rapid HIV/2 ADVANCED QUALITY Rapid HIV Test MedMira Rapid HIV Test First Response HIV/HIV2 WB CAPILLUS HIV /HIV2 UniGold HIV Detection After Reference Assay average 9% confidence limits

26 Explanatory notes for Tables 6 and Figure Table General characteristics and operational aspects of the assays. Specimen type and volume All seven assays under evaluation may be used with human whole blood, serum or plasma samples. The volume of whole blood required for each assay ranged from µl to 6µl. Shelf life at ( C) Only one kit, the Capillus HIV/HIV2 required storage at 2 8 C, the remaining six kits could be stored at room temperature. Final dilution of the serum is the dilution of the serum in the test format, e.g. µl serum added to 2µl diluent gives a final dilution of /2. Total time to perform the assay Price/test Table 2 Sensitivity Specificity reflects the time needed to carry out test The indicative catalogue price, in US $, as given by the manufacturer, or converted to USD using the currency conversion rate at the time. Comparison of the results of the assays with reference tests calculated as described on page 8 of this document. calculated as described on page 8 of this document. 9% Confidence limits (CL) calculated as described on page 9 of this document PPV and NPV Indeterminate results Interreader variability Table Number of specimens per run Minimum maximum calculated as described on page 9 of this document Simple/rapid assays test results which could not be interpreted as clearly positive or negative were considered indeterminate. calculated as described on page of this document. Detailed operational aspects of the assay minimum number = sample in addition to the required controls maximum number = the maximum number of samples in addition to the required controls which can be simultaneously tested within the limits of the assay procedure. Page 2

27 Table cont. Number of controls per test run Internal control Definition of positive results Tables 4a and 4b Table Note Table 6 Figure Detailed operational aspects of the assay Explanatory notes for Tables 6 and Figure The InstantScreen Rapid HIV/2 assay (GAIFAR) has a control spot on the test devices which checks that sample has been added, the procedure is followed correctly and reagents function correctly. The Capillus HIV/HIV2 (Trinity Biotech) assays contains no internal control. The original MedMira HIV Rapid Test provided for the evaluation contained no internal control, however a futher version was supplied for the seroconversion panels which contained a procedural control line. The remaining four assays have a control spot or line on the test devices which checks that reagents function correctly and the procedure has been correctly followed. a sample is interpreted as positive according to the criteria set by the manufacturer and summarised in the table Calculation of ease of performance of the assay The criteria for this calculation are given in the respective tables. Suitability of the assay for use in small laboratories The criteria for this calculation are given in the respective table. These criteria are primarily technical and while an assay may be regarded as technically suitable for use in laboratories with limited facilities or where small numbers of samples are routinely tested, the sensitivity and specificity of the assay are overriding factors in determining the suitability of an assay for use in any laboratory. Performance of the assays on seroconversion panels An assay s performance on the seroconversion panels should be viewed against both the sensitivity and specificity of the assay. Assays of relatively low specificity may appear to detect antibody to HIV earlier than other assays of higher specificity. Caution should be taken when reviewing seroconversion performance of assays tested only in 8 panels. Relative performance on seroconversion panels Eight seroconversion panels (BBI), each containing several samples taken at different time intervals early in the infection period (window period), were tested with the simple and/or rapid antihiv test kits. The results obtained with these assays were compared with those of the combined outcome of the Enzygnost AntiHIV /2 Plus reference test (see page 7 of this report); the difference in the relative position in the sample sequence for the first sample of a panel to become positive with test X as compared with the first positive result with the reference test. If a test gave a positive result earlier than the reference test the difference in detection was rated as negative; if the test became positive later the number was rated as positive. If the assay detected no specimens in the panel then the assigned value was total number of specimens in the panel. The mean of the difference in the number of specimens for a test to become positive as compared to the reference test was calculated and plotted on a yardstick. The 9% confidence limits of the mean were also calculated. These limits should be interpreted with caution as only 8 panels were tested. Page 22

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29 WHO/BCT/2.7 Page 2 6 ANNEXES ANNEX. Algorithm for characterisation of the WHO HIV whole blood matched serum specimen panel EIA Vironostika HIV Unifrom II plus (Organon Teknika) EIA 2: Enzygnost AntiHIV /2 Plus, (Dade Behring) / repeat ELISAS / INNOLIA HIV Confirmation POS IND NEG HIV HIV2 dual reactive further investigation: HIV Ag Western Blot PCR PEPTILAV 2 Anti HIV POS INDETERMINATE Anti HIV NEG

30 WHO/BCT/2.7 Page 24 ANNEX 2. List of assay manufacturers /distributors addresses Abbott Laboratories,. Diagnostics Division, Abbott Park Road, Abbott Park, Il 664, USA. Tel: ; Fax: ; Website: GAIFAR GmbH, Biotech Campus Hermannswerder 6, 447 Potsdam, Germany. Tel: ; Fax ; Website: Intec Products Inc. (Xiamen), 2 Xinguang Road, Xin Yang Industrial Area, Haicang, Xiamen, China, 622. Tel: ; Fax: ; Website: PMC Medical (India) Pvt. Ltd., Gala No. 2, Shree Ganesh Industrial Estate, Kachigam, NaniDaman, Daman962, India. Tel: ; Fax: ; Website: MedMira Laboratories Inc. 2 Ronson Drive, Suite, Toronto, Ontario, M9W Z9. Tel: ; Fax: ; Website: Trinity Biotech PLC, IDA Business Park, Bray, Co. Wicklow, Ireland. Tel: ; Fax: ; Website: 7 ADDITIONAL READING Additional reading may be obtained by visiting the BCT section of the WHO website at and follow the links to Key Initiatives and HIV Diagnostics. In addition to general information on diagnostics, assay evaluation reports for HIV, HCV and HBV are available as well as details for the WHO HIV Test Kit Bulk Procurement Scheme. 8 ACKNOWLEDGEMENTS We should like to thank the Institute of Tropical Medicine, Antwerp, Belgium for supplying the prospectively collected whole blood and matched serum samples that constituted the evaluation panel for this evaluation. We acknowledge the six companies, Abbott Laboratories, GAIFAR GmbH, Intec Products Inc., PMC Medical Pvt. Ltd., MedMira Laboratories Inc. and Trinity Biotech PLC for supplying the test kits free of charge. 9 NOTE Other HIV tests using whole blood were available at the time of this evaluation but, due to the nature, design and capacity of this prospective study, it was not possible to include them in the evaluation.