HIV ASSAYS: OPERATIONAL CHARACTERISTICS

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HIV ASSAYS:

OPERATIONAL CHARACTERISTICS

(PHASE I)

REPORT 12

SIMPLE/RAPID TESTS

WHOLE BLOOD SPECIMENS

World Health

Organization

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HIV ASSAYS:

OPERATIONAL CHARACTERISTICS

(PHASE I)

REPORT 12

SIMPLE/RAPID TESTS

WHOLE BLOOD SPECIMEN

World Health Organization

Department of Essential Health Technologies

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WHO Library Cataloguing-in-Publication Data World Health Organization.

HIV assays : operational characteristics (Phase I). Report 12: simple/rapid tests, whole blood specimens.

1.AIDS serodiagnosis - methods 2.Blood - virology 3.Reagent kits, Diagnostic- utilization 4.Clinical trials, Phase I 5.HIV seropositivity - diagnosis I.Title.

ISBN 92 4 159235 4 (NLM classification: WC 503.1) This publication is a reprint of material originally distributed as WHO/BCT/02.07

© World Health Organization 2002

All rights reserved. Publications of the World Health Organization can be obtained from Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 2476; fax: +41 22 791 4857; email: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to Marketing and Dissemination, at the above address (fax: +41 22 791 4806; email: permissions@who.int).

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.

The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

WHO, UNAIDS, and the Institute of Tropical Medicine, Antwerp, Belgium, do not warrant or represent that the evaluations conducted with the HIV test kits referred to in this document are accurate, complete and/or error-free. WHO, UNAIDS and the Institute of Tropical Medicine disclaim all responsibility for any use made of the data contained herein, and shall not be liable for any damages incurred as a result of its use. This document must not be used in conjunction with commercial or promotional purposes.

Printed in France

_____________________________________________________________________

Contact: Dr G. Vercauteren, Essential Health Technologies - WHO - 20, Avenue Appia - 1211 Geneva 27- Switzerland

This document is available on the internet at: www.who.int/eht/

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HIV ASSAYS: OPERATIONAL CHARACTERISTICS (PHASE I) REPORT 12

SIMPLE/RAPID TESTS WHOLE BLOOD SPECIMENS Table of Contents

1. Introduction 1

2. Background information 1

3. Laboratory aspects of HIV testing 3

3.1 A brief overview 3

3.2 Quality assurance 3

3.3 Safety 4

4. Materials and Methods 4

4.1 Assays (test kits) evaluated 4

4.2 Evaluation panel 8

4.3 Laboratory testing 8

4.4 Data analysis 9

4.4.1 Sensitivity, specificity, confidence limits and predictive values

of HIV tests 9

4.4.2 Inter-reader variability 10

4.4.3 Sensitivity in seroconversion panels 10

4.4.4 Additional analyses 11

5. Assay evaluations 11

Table 1: General characteristics and operational aspects 13 Table 2: Comparison of whole blood anti-HIV assays with reference tests 14

Table 3: Detailed operational aspects 15

Table 4a: Technician's appraisal of the test kits 17

Table 4b: Calculation of ease of performance 18

Table 5: Suitability for use in small laboratories 19

Table 6: Performance on seroconversion panels 20

Figure 1: Relative performance on seroconversion panels 22

Explanatory notes for Tables 1-6 and Figure 1 23

6. Annexes 25

Annex 1: Algorithm for characterization of the WHO HIV panel 25 Annex 2: List of assay manufacturers'/distributors' addresses 26

7. Additional reading 26

8. Acknowledgements 26

9. Note 26

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1 I

NTRODUCTION

Since 1988, 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 decision-makers 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, shelf-life and ease of use are also provided.

Report 12, 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 2000 and September 2000:

• Determine™ HIV-1/2 (Abbott Laboratories)

• InstantScreen™ Rapid HIV-1/2 Assay (GAIFAR GmbH)

• ADVANCED QUALITY™ Rapid HIV Test (InTec Products Inc.)

• MedMira Rapid HIV Test (MedMira Laboratories Inc.)

• First Response™ HIV-1/HIV-2 WB (PMC Medical Pty. Ltd)

• CAPILLUS™ HIV-1/HIV-2 (Trinity Biotech PLC)

• Uni-Gold™ HIV (Trinity Biotech PLC)

This report provides data of phase I of the WHO coordinated evaluation of seven anti- HIV simple/rapid (S/R) test kits using whole blood specimens. Phase II, is planned for 2002, 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, 1211 Geneva 27, Switzerland. The report is also available on the BCT section of the WHO website www.who.int/bct

2. B

^ACKGROUND

In the last few years there have been a number of improvements of S/R tests for the detection of HIV antibodies (anti-HIV). 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 counselling and testing (VCT) and/or prevention of mother-to-child 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,

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whether carried out in central 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 anti-HIV 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 15 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 counselling and testing services (VCT).. 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 30°C, some even up to 45°C. In most cases the kits contain the reagents ready-for-use 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 resource-poor 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

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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 cost-effective, and possibly preferable, in many situations.

3. L

ABORATORY ASPECTS OF

HIV

TESTING

3.1 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 antibody-positive 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 1 and/or HIV 2 proteins, have been developed, eg INNOLIA and Pepti-LAV. 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.

3.2 Quality assurance

All laboratories carrying out HIV tests, should have a well-functioning 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

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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.

3.3 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, 1993 (ISBN 92 4 154450 3) and the Communicable Diseases Surveillance and Response section of the WHO website, www.who.int/emc , where information on laboratory biosafety and transport of infectious substances may be found.

4 M

ATERIALS AND

M

ETHODS

4.1 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 3.2).

Determine™ HIV-1/2 (Abbott Laboratories, Il, USA) Product code 7D23-33

An in-vitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV 1 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.

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InstantScreen™ Rapid HIV-1/2 Assay (GAIFAR GmbH, Potsdam, Germany) Product code not available

An in-vitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV 1 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-1/2 assay Generation 1 and InstantScreen™ Rapid HIV-1/2 assay Generation 2. The assays differ in formulation, binding procedure and concentrations of the antigen components of the assay. The Generation 1 assay has been replaced by the Generation 2 assay.

(Image of the test device is not available)

ADVANCED QUALITY™ Rapid HIV Test (InTec Products Inc., Xiamen, China) Product code ITP02002-TC40

An enhanced rapid immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV 1 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 20 seconds before addition of the sample diluent.

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MedMira Rapid HIV Test (MedMira Laboratories Inc. Toronto, Canada) Product code not available

An in-vitro immunofiltration test for the qualitative detection of antibodies to HIV 1 and HIV 2 in human serum, plasma 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 1, was tested in the WHO whole blood panel. The 2-line format, version 2, was a later version of the assay which was used for testing with the commercial seroconversion panels.

First Response™ HIV-1/HIV-2 WB (PMC Medical Pty. Ltd., Daman, India) Product code DD/138

An in-vitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV 1 and HIV 2 in human serum, plasma and whole blood. 25µl of serum/plasma or 30µ l whole blood is required for the assay, otherwise the testing procedure is the same for all specimen types.

Version 1 Version 2

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CAPILLUS™ HIV-1/HIV-2 (Trinity Biotech PLC., Bray, Ireland) Product code 6048G

A latex agglutination test for the detection of antibodies to HIV 1 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.

Uni-Gold™ HIV (Trinity Biotech PLC., Bray, Ireland) Product code 1206502

An in-vitro immunochromatographic (lateral flow) test for the qualitative detection of antibodies to HIV 1 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 Uni-Gold™

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 Uni-Gold™ HIV peptide test is no longer in production.

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4.2 Evaluation panel

Two hundred and fifty (250) 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 anti-HIV positive and 170 anti-HIV 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 1

Anti-HIV Positive Anti-HIV Negative Total

80 170 250

32% 68% 100%

For characterization, the matched serum samples were screened with 2 reference ELISAs, Vironostika HIV Uni-Form II plus O (Organon Teknika) and the Enzygnost Anti-HIV 1/2 Plus (Behringwerke AG). Specimens non-reactive in both assays were considered anti-HIV negative. Specimens that 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 1.

In addition to the anti-HIV positive and negative samples, eight commercial serum/plasma seroconversion panels from Boston Biomedica Inc. (BBI) were tested in each of the 7 assays evaluated.

4.3 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 3 times were recorded as the final results. Samples from the commercial serum/plasma seroconversion panels giving discordant results were not retested.

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4.4 Data analysis

4.4.1 Sensitivity, specificity, confidence limits (CL) and predictive values of anti-HIV 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 anti-HIV status

+ –

Results of assay + a

True-positives

b False-positives

a+b under

evaluation – c

False-negatives

d True-negatives

c+d

a+c b+d

Sensitivity = a/(a+c) Positive predictive value = a/(a+b) Specificity = d/(b+d) Negative predictive value = d/(c+d)

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 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 95% confidence limits are a means of determining whether observed differences in sensitivity or specificity between assays are significant or not. Exact 95% confidence limits for Binomial proportions were calculated from the F-distribution (Armitage P. and Berry G. Statistical Methods in Medical Research, 2^nd Edition. Blackwell Scientific Publications, Oxford, 1987, page 119).

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:

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1. using the simple formula a/(a+b) 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

(prevalence)(sensitivity)

PPV= ____________________________________________

(prevalence)(sensitivity) + (1 - prevalence)(1 - 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:

1. the simple formula d/(c+d) 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:

(1 - prevalence)(specificity)

NPV= _____________________________________________

(1 - prevalence)(specificity) + (prevalence)(1 - 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 false-positive 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 false-negative. However, this effect only becomes apparent at prevalence of 80% and above.

4.4.2 Inter-reader variability

The inter-reader 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.

4.4.3 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 Anti- HIV 1/2 Plus (Behringwerke AG), the assay arbitrarily designated the reference for determination of relative sensitivity in these panels. For each seroconversion series

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(panel) the first specimen in the sample sequence to become reactive with Enzygnost Anti-HIV 1/2 Plus (Behringwerke AG) was assigned the value “0”. Results from the assays under evaluation were compared with Enzygnost Anti-HIV 1/2 Plus (Behringwerke AG) by determining the difference between the specimen assigned value“0” 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 Anti-HIV 1/2 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 1/2 Plus (Behringwerke AG), the value assigned was +1. If no specimens in the panel are positive then the assigned value was 1+ 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 95% confidence limits were determined. The results for the seroconversion panels are presented in Table 6 and diagrammatically in figure 1.

4.4.4 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.

5. A

SSAY EVALUATIONS

Results from the testing of the HIV whole blood and seroconversion panels are presented in Tables 2 and 6 and figure 1 on the following pages. Of the seven assays, six obtained 100% 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=80) 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 100%, and the other 3 obtained 97.6%, 98.8% and 99.4% respectively.

The results for both sensitivity and specificity should be interpreted carefully and by taking the 95% confidence limits (95%CL) into account.

Many of these assays have been evaluated in serum/plasma panels previously by WHO and other groups. The Determine HIV-1/2 (Abbott), Capillus HIV-1/2 (Trinity Biotech) and UniGold HIV (Trinity Biotech) have been evaluated on serum/plasma panels previously by WHO. This information is available on the WHO website www.who.int/bct . However, the focus of this evaluation was specific in assessing the performance of these 7 S/R anti-HIV tests on whole blood samples.

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ASSAY EVALUATIONS

Whole Blood Specimens

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Table 1. General characteristics and operational aspects

Name Determine™

HIV-1/2

InstantScreen™

Rapid HIV-1/2 Assay*

ADVANCED QUALITY™

Rapid HIV Test

MedMira Rapid HIV

Test

First Response™

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™ HIV

Company Abbott Laboratories,

Dainabot Co. Ltd., Tokyo, Japan

GAIFAR GmbH, Potsdam, Germany

InTec Products Inc., Xiamen, China

MedMira Laboratories Inc., Halifax, Canada

PMC Medical Pty.

Ltd., Daman, India

Trinity Biotech PLC,

Bray, Ireland

Trinity Biotech PLC, Bray, Ireland

Assay type immunochromatographic immunofiltration immunochromatographic immunofiltration immunochromatographic agglutination immunochromatographic

Solid phase membrane membrane membrane membrane membrane polystyrene latex

beads

membrane

Antigen type recombinant/synthetic epitope-combi- antigens

recombinant synthetic recombinant recombinant recombinant

Specimen type optional

whole blood serum/plasma

whole blood serum/plasma Number of tests per kit

(product code)

100 (7D23-33)

20 (not available)

40 (ITP02002-TC40)

50 (not available)

35 (DD/138)

100 (6048G)

20 (1206502) Lot numbers evaluated

(expiry date)

62509U102 (13/4/2001)

Gen 1: 01199 (12/2000) Gen 1: ISO1499

(12/2000) Gen 2: ISO 1700

(01/2001)

RD 0907019 (10/2001)

CTIA 0009 (5/2001)

300D12 (8/2001)

G35412 (13/3/2001)

G32307 (03/02/2001)

Shelf life ( at °C)

18 mths (2 – 30)

18 mths (-20 to +45)

18 mths (2 – 30)

1 year (2 – 30)

18 mths (2 – 30)

20 mths (2 – 8)

20 mths (2 – 27) Volume of whole blood needed

(µl)

Final dilution of whole blood

50

none

20

none

5

1/21

≈ 30 none

30

none

10

1/13

≈ 60

none Total time to perform the assay:

h.min. (1 specimen)

0.17 0.03 0.01 0.05 0.01 0.03 0.11

Reading visual visual visual visual visual visual and/or by

Capillus Digital Reader

visual

Price/test (US$) 1.20 8 - 12 0.80 – 1.20 3 1.15 2.20 2.34

*Two versions of the InstantScreen assay were evaluated, Generation 1 and Generation 2.

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Table 2. Comparison of the whole blood HIV assays with reference test

HIV-1/2

Rapid HIV-1/2 Assay*

Rapid HIV Test

MedMira Rapid HIV Test

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™ HIV

Final sensitivity % (95 CL)¹ n=80

100.0 (95.5 – 100.0) Gen 1: n=46 98.0 (89.0 – 100.0)

Gen 2: n=34 100.0 (90.0 – 100.0)

98.8 (93.2 – 100.0) 100.0 (95.5 – 100.0) 100 (95.5 – 100) 100.0 (95.5 – 100.0) 100.0 (95.5 – 100.0)

Initial specificity % (95 CL)

Final specificity % (95 CL) n=170

99.4 (96.7 – 100.0)

Gen 1: n= 62 98.0 (91.0 – 100.0)

Gen 2: n=108 99.0 (95.0 – 100.0)

Gen 1: n= 62 98.0 (91.0 – 100.0)

Gen 2: n=108 100.0 (97.0 – 100.0)

97.5 (91.3 – 99.7)

100.0 (97.9 – 100.0)

91.2 (85.9 – 95.0)

97.6 (94.1 – 99.6)

98.2 (94.9 – 99.6)

98.8 (95.8 – 99.9)

100.0 (97.9 – 100.0)

Indeterminate results % 0.0 Gen 1: 0.0

Gen 2: 0.0

0.8 0.8 0.0 0.0 0.0

Initial inter-reader variability % 1.6 Gen 1: 0.0 Gen 2: 0.7

2.0 14.4 0.4 0.0 0.4

PPV 0.1%²

6.0%

1.64²

91.41

Gen 1: 0.05 Gen 2:100.0 Gen 1: 75.8 Gen 2: 100.0

100.0

0.41²

72.67

0.83²

84.18

100.0

NPV 0.1%

6.0%

100.0

Gen 1: 100.0 Gen 2: 100.0 Gen 1: 99.9 Gen 2: 100.0

100.0

99.9

100.0

195% 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 34 HIV positive specimens of the whole blood panel were tested in the Generation 1 and Generation 2 InstantScreen assays, respectively, and 62 and 108 HIV negative specimens were tested in the Generation 1 and Generation 2 assays, respectively.

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Table 3. Detailed operational aspects

HIV-1/2

Rapid HIV-1/2 Assay

Rapid HIV Test

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™ HIV

Dimension (cm) of kit: w-l-h 27 - 16 -1 30 - 19 - 9 22 - 13.5 - 9 37.5 - 27.5 - 9.5 21 - 12.3 - 7 22 - 16.5 - 6.5 23 - 14 - 10.5

Storage conditions (°C) 2 - 30 -20 – +45 2 - 30 2 - 30 2 - 30 2 - 8 2 - 27

Incubation temperature (°C) room temperature none stated room temperature 20 - 30 room temperature room temperature room temperature

Reading endpoint stability (h.min)

1.0 >24.00 0.01 - 0.20 0.15 0.10 0.30 0.20

Stability after dilution/

Reconstitution/opening (°C) - antigen

- controls - sample diluent - conjugate - substrate - wash buffer

expiry date (2 - 30) not applicable not applicable expiry date (2 - 30)

not applicable expiry date (2 - 30)

expiry date (-20 – +45) not applicable expiry date (-20 – +45) expiry date (-20 – +45)

not applicable expiry date (-20 – +45)

expiry date (2 - 30) not applicable expiry date (2 - 30)

not applicable not applicable not applicable

expiry date (2 - 30) 1 week (2 - 8) not applicable 1 week (2 - 8) not applicable expiry date (2 - 8)

expiry date (2 - 30) not applicable not applicable not applicable expiry date (2 - 30)

not applicable

expiry date (2 - 8) expiry date (2 - 8) not applicable not applicable not applicable not applicable

expiry date (2 - 27) not applicable not applicable not applicable not applicable expiry date (2 - 27) Number of sera per run

Minimum - maximum 1 - 10 1 - 5 1 - 5 1 - 5 1 - 5 1 - 10 1 - 10

Number of controls per test run - negative

- cut-off/weak positive - positive

- blank

internal control: reagent control : sample addition

control

0 0 0 0

1 0

0 0 0 0

0 1

0 0 0 0

1 0

1 0 1 0

0*

0

0 0 0 0

0 1

1 0 1 0

0 0

0 0 0 0

1 0

*

Version 1 of the MedMira HIV Rapid test does not contain a reagent control. The later version 2 of the assay contains a reagent control line.

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Table 3. (continued) Detailed operational aspects

HIV-1/2

Rapid HIV Test

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™ HIV

Estimated time to perform one run:

h. min

0.17 0.03 0.01 0.05 0.01 0.03 0.11

Equipment needed but not provided in the kit¹:

- washer

- incubator (water-bath) - spectrophotometric reader - refrigerator (storage) - agitator, rocker - aspiration device - automatic pipette (µl) - multichannel pipette (µl) - 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 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 pink- purple test and

control lines

Appearance of any agglutination

Appearance of test and control lines

Definition of grey zone or Indeterminate result

Not applicable Not applicable Not applicable Not applicable Not applicable * Not applicable

1+: 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.

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Table 4a. Technician's appraisal of the test kit

Name Score Determine™

HIV-1/2

Rapid HIV Test

Test

First Response™

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™

HIV

Number of steps in the test procedure:

- 1-2 steps - 3-5 steps - >5 steps

6 3 1

6 3 6 1 6 6 6

Clarity of kit instructions:

- good

- needs improvement

2 1

2 1 1 2 2 2 2

Kit and reagent packaging and labelling:

- good

- needs improvement

2 1

2 2 2 2 1 2 2

Total (out of possible 10) 10 10 6 9 5 9 10 10

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 20

minutes.

Red cells observed after removal of

pre-filter for 5 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.

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Table 4b. Calculation of ease of performance

HIV-1/2

Rapid HIV-1/2 Assay (Gen2)

Rapid HIV Test

Test

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™

HIV Need to prepare:

- antigen - substrate - wash solution - conjugate

- predilution of serum

1¹ 1 1 1 1

1 1 1 1 0²

1 1 1 1 1

1 1 1 0 1

1 1 1 1 1

1 1 1 1 1 Stability after dilution/opening:

(expiry date = 1; less = 0) - antigen

- controls - sample diluent - conjugate - substrate - wash buffer

1 1 1 1 1 1

1 0 1 0 1 1

1 1 1 1 1 1

1 1 1 1 1 1 Sufficient reagents

- wash buffer (yes = 1; no = 0)

1 1

1 1 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

1 1 1 1 1 1 1

1 0 1 1 1 1 1

1 1 1 1 1 1 1

1 0 1 1 1 1 1

1 1 1 1 1 1 1

1 1 1 1 1 1 1 Technician's appraisal of the test kit³

(rating out of 10)

10 6 9 5 9 10 10

Total (out of possible 30) 30 25 28 22 28 30 30

Ease of performance:

- less easy <20 - easy 20 # x # 25 - very easy >25

very easy very easy very easy easy very easy very easy very easy

11 : positive rating: reagent needs no preparation; item provided in the kit ²0 : negative rating: reagenàpt needs preparation; item not provided in the kit ³: see table 4a

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Table 5. Suitability for use in small laboratories

Name Score Determine™

HIV-1/2

Rapid HIV Test

Test

HIV-1/HIV-2 WB

CAPILLUS™

HIV-1/HIV-2

Uni-Gold™

HIV

Sensitivity - 100%

- 98 - 100%

- <98%

Specificity - >98%

- 95 - 98%

-<95%

Incubation temperature - room t°

- other than room t°

Shelf-life - >1 year

- ∃6 months # 1 year - < 6 months Storage at

- ambient t° possible (opened kit) - ambient t° possible (unopened kit) - 2-8°C required

Price per test (US$) - #1.0 - #2.0 - > 2.0 Ease of performance - very easy - easy - less easy

Rapidity of performance: 1 serum - < 10 min

- 10 – 30 min - > 30 min Washer/agitator - not needed - needed Reading

- visual: inter-reader variability #3%

: inter-reader variability >3%

- reading equipment

5 3 0

3 1

3 2 1

5 2 1

3 2 1

5 3 1

3 2 1

3 1

5 3 1

5

3

5

2

5

2

3

5

3

5

1

5

3

5

3

5

2

5

3

5

3

5

1

3

5

3

5

2

5

3

5

3

1

5

3

5

3

5

1

5

2

3

5

Total (out of possible 40) 38 38 35 32 39 34 37

Suitability for use in small laboratories:

- less suitable < 23 - suitable # x # 30

- very suitable > 30

very suitable very suitable very suitable very suitable very suitable very suitable very suitable Page 19