Draft Report WHO evaluation of HIV 1/2 STAT-PAK (Chembio Diagnostic Systems Inc)

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1 Date: 6 January 2012 Draft Report WHO evaluation of HIV 1/2 STAT-PAK (Chembio Diagnostic Systems Inc) WHO Collaborating Centre for HIV/AIDS Diagnostic and Laboratory Support Department of Clinical Sciences Institute of Tropical Medicine Antwerp, Belgium World Health Organization Geneva, Switzerland PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 1 of 27

2 Table of Contents 1. Details of the product Name of product Manufacturer Indicative price per test and product codes General information about the product Type (format) of assay Type of antigen Type of solid phase Contents of test kit Test kit controls Conjugate (and diluent) Substrate (and diluent) Test kit dimensions (width-length-height) Labels Quantity of reagents Instructions for use Storage conditions Shelf life Operational aspects of the product Reagents Stability after opening/preparation ( C) Specimens Specimen type Specimen volume Number of specimens per run Incubation temperature Washing procedure Reading procedure Time required to perform the assay (h:min) Equipment and consumables required but not provided in the test kit Materials and methods Specimens WHO HIV specimen reference panel Batch-to-batch variation panel HIV seroconversion panels HIV mixed titer panel WHO reference preparations External quality control specimen Reference results Batch numbers and expiry dates of test kits Interpretation of testing results Endpoint stability Run validation Data analysis Performance characteristics Sensitivity Specificity Confidence intervals Predictive values Indeterminate results Discrepant results Interpretation of results from batch-to-batch panel Interpretation of results from HIV seroconversion panels Interpretation of results from HIV mixed titer panel Interpretation of results from WHO reference preparations Inter-reader variability Results Validation of results Comparison of initial testing results with reference results Detection of antibodies to HIV-1/ Comparison of final testing results with reference results...13 PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 2 of 27

3 Detection of antibodies to HIV-1/ Discrepant results Predictive values Results from batch-to-batch panel Results from HIV seroconversion panels Results from HIV mixed titer panel Results from WHO reference preparations Inter-reader variability Appraisal by laboratory technician Summary References...17 PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 3 of 27

4 1. Details of the product 1.1. Name of product HIV 1/2 STAT-PAK A qualitative screening test kit for the detection of antibodies to HIV-1/2 in human fingerstick and venous whole blood, and serum and plasma specimens Manufacturer Chembio Diagnostics Systems, Inc Horseblock road Medford, NY United States Tel: Fax: Corporate web site: Indicative price per test and product codes Product code No. of tests per kit Indicative price in operating currency HIV US$ 1.50 HIV104 (HIV Control Set)* HIV-1 reactive, HIV-2 reactive, HIV negative N/A *HIV Control Set was requested for this evaluation but not submitted. 2. General information about the product 2.1. Type (format) of assay Lateral flow immunochromatographic assay 2.2. Type of antigen HIV 1/2 proteins 2.3. Type of solid phase Nitrocellulose membrane 2.4. Contents of test kit The product is available in pack sizes of 20 tests. For this performance evaluation, the 20 test pack size was evaluated. Each test kit contains individually pouched test devices, running buffer (3.5ml), disposable sample loops (5µl) and instructions for use (product insert) Test kit controls Test kit controls: Test kit controls are not supplied in the test kit but available on order from the manufacturer. Each package contains: HIV-1 reactive control, HIV-2 reactive control, non-reactive control and instructions for use (product insert). Specimen addition control: The control line only appears if human specimen is added to the device and is therefore a specimen addition control line. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 4 of 27

5 2.6. Conjugate (and diluent) Conjugate: colloidal gold conjugated to a specific antibody binding protein. Conjugate diluent: not applicable Substrate (and diluent) Substrate: not applicable. Substrate diluent: not applicable Test kit dimensions (width-length-height) The dimension of the pack size of 20 tests is 16.5 / 13 / 7 cms Labels All reagents and devices were clearly labelled. The batch number and expiry date were visible on the outer packaging and individual components including the pouch containing the test device and running buffer bottle. The test cassette itself is not identified with the product name, batch number and expiry date Quantity of reagents All reagents were supplied in sufficient quantities for the serum/plasma test procedure Instructions for use The instructions for use (package insert) were clear. Aspects of the performance of the test procedure including specimen collection, specimen storage, reading time, endpoint stability time, and result interpretation were clear Storage conditions The product must be stored at 8-30 C Shelf life According to the manufacturer, the shelf life of the product upon manufacture is 24 months. 3. Operational aspects of the product 3.1. Reagents No preparation of reagents was required Stability after opening/preparation ( C) Unopened test devices and running buffer are stable until the expiry date when stored at 8-30 C Specimens Specimen type This product is intended for use with serum/plasma, and venous/capillary whole blood specimens. For this performance evaluation, serum/plasma specimens were used. Venous whole blood must be collected using tubes containing citrate, heparin or EDTA as anticoagulant. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 5 of 27

6 Specimen volume 5 µl of specimen was required to perform the assay. A sample loop to add the specimens is supplied within the kit. For this evaluation, the specimens were added by the use of the sample loops Number of specimens per run Minimum number of specimens per run: 1 Maximum number of specimens per run: Incubation temperature The incubation temperature should be between 18 to 30 C, for this evaluation all testing was conducted between C Washing procedure Not applicable Reading procedure The results were read visually according to the instructions for use. In addition, the intensity of each test band was scored and entered into the data collection sheet Time required to perform the assay (h:min) Time to test one specimen: 0:17 Time to test one run (10 specimens): 0: Equipment and consumables required but not provided in the test kit The following equipment were required but not provided: timer, refrigerator for storage of test kits where recommended storage temperature could not be provided (facultative). The following consumables were required but not provided: For capillary whole blood specimens: alcohol swabs and lancets. For venous whole blood and serum/plasma specimens: blood collection equipment. 4. Materials and methods 4.1. Specimens WHO HIV specimen reference panel The panel consisted of approximately 1079 clinically derived serum/plasma specimens of European, African, Latin America and Asian origin, see Table 1. There were 421 anti-hiv positive specimens, of which 16 are anti-hiv-2 positive and 658 anti-hiv negative specimens. Table 1 - WHO Specimen Evaluation Panel HIV positive specimens HIV negative specimens Total number Batch-to-batch variation panel Batch to batch variation is assessed using a panel of ten anti-hiv positive specimens diluted in 2-fold dilutions to make 16 member dilution series (n=160) on two separate production batches. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 6 of 27

7 HIV seroconversion panels Eight HIV seroconversion panels: PRB914, PRB925, PRB926, PRB930, PRB955, PRB965, PRB968, PRB 969 (sourced from SeraCare Life Science Inc) were tested in singular on one production batch HIV mixed titer panel One anti-hiv mixed titer performance panel containing 25 members: PRB205 (sourced from SeraCare Life Science Inc) was tested in singular on one production batch WHO reference preparations The WHO international biological reference preparation panel with the catalogue number 02/210 (Anti-HIV antibodies [HIV-1 subtypes A, B, C, CRF01_AE, group O and HIV-2] were tested in singular on one production batch External quality control specimen An appropriate external quality control specimen was testing during each testing session. The QC specimen was produced by ITM according to their procedures Reference results The WHO HIV specimen reference panel was characterized according to a standard combination of assays i.e. a standardized testing algorithm. These reference testing results were used to determine the true HIV status of each specimen for the purpose of this performance evaluation, see Figure 1. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 7 of 27

8 EIA 1 Vironostika HIV Ag/Ab (biomérieux) EIA 2 Enzygnost Anti-HIV 1/2 (Siemens Healthcare Diagnostics) in parallel Anti-HIV Negative INNO-LIA HIV I/II Score line immunoassay (Innogenetics) Positive Indeterminate Negative Anti-HIV-1 Positive Anti-HIV Positive Anti-HIV-2 Positive INNOTEST HIV Antigen mab (Innogenetics) INNOTEST HIV Antigen mab (Innogenetics) New LAV II Blot (BioRad Laboratories) Neutralisable Ag Non neutralisable Ag Non neutralisable Ag Neutralisable Ag Positive Anti-HIV Positive Indeterminate Negative Anti-HIV Positive HIV Antigen Positive/ Anti-HIV inconclusive Excluded from the analysis if evaluation of 3rd gen assay but included if evaluation of 4th gen assay but considered as a true positive specimen Indeterminate - excluded from panel Anti-HIV Negative HIV Antigen Positive Excluded from the analysis if evaluation of 3rd gen assay but included if evaluation of 4th gen assay but considered as a true positive specimen PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 8 of 27

9 Initially, each specimen was tested on the Vironostika HIV Ag/Ab EIA (biomérieux) and Enzygnost Anti-HIV 1/2 Plus EIA (Siemens Healthcare Diagnostics) in parallel. Specimens that are non-reactive on both EIAs were assigned anti-hiv negative. Specimens with discrepant EIA results AND with dually reactive results on both EIAs were tested on the INNO-LIA HIV I/II Score line immunoassay (Innogenetics). Specimens that were negative by line immunoassay were further tested on Innotest HIV antigen EIA (Innogenetics) and if found non-reactive then were assigned anti-hiv negative. If found to be neutralisable for HIV-1 antigen, the specimen was considered HIV-1 antigen positive and anti- HIV negative and was retained for the evaluation of 4 th generation assay but not for 3 rd generation assays. Specimens that are indeterminate by line immunoassay were further tested on Innotest HIV antigen EIA (Innogenetics) and if found non-reactive then were excluded from the panel. Specimens that were reactive for antigen (and were neutralisable) were assigned as HIV-1 antigen positive and anti-hiv inconclusive. These specimens were retained for the evaluation of 4 th generation assay but not for 3 rd generation assays. Specimens that were positive by line immunoassay were assigned as anti-hiv-1 positive or anti-hiv-2 positive. Those specimens that could not be discriminated (i.e. anti-hiv positive) were further tested on the New LAV II Blot (BioRad Laboratories). Specimens that were indeterminate or negative by the New LAV II Blot were assigned as anti-hiv-1 positive. Specimens that were positive by the New LAV II Blot were assigned as anti-hiv positive. All reference assays were interpreted according to IFU as given by the manufacturer. The data obtained with HIV 1/2 STAT-PAK were compared to the reference testing results. All data from reference testing is shown together with the results of the HIV 1/2 STAT-PAK results in Annexes 1 and Batch numbers and expiry dates of test kits Batch number A: Expiry date: 02/02/2013 Batch number B: Expiry date: 26/04/ Interpretation of testing results In this performance evaluation, 10 specimens were tested per run. The test procedure was performed by one laboratory technician; the results were read independently by three technicians Endpoint stability The reading endpoint is stated, for this performance evaluation the results were read 15 minutes after the addition of specimen to the test device but no longer than 20 minutes Run validation The criteria for run validation were in accordance with the instructions for use. The pink/purple line appeared in the control area of all tests. 5. Data analysis 5.1. Performance characteristics The following methods were used to calculate the performance characteristics, see Table 2. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 9 of 27

10 Table 2-2x2 table for calculation of performance characteristics Results of assay under evaluation Reference testing results a b true positives false positives - c d false negatives true negatives a + c b + d a + b c + d Sensitivity Sensitivity is the ability of the product under evaluation to detect correctly sera that contain antibody to HIV (reference assays positive). Thus sensitivity is the number of true positive sera identified by the product under evaluation as positive (a), divided by the number of sera identified by the reference assays as positive (a+c), expressed as a percentage. Sensitivity = a a + c Specificity Specificity is the ability of the product under evaluation to detect correctly sera that do not contain antibody to HIV (reference assays negative). Thus specificity is the number of true negative sera identified by the product under evaluation as negative (d), divided by the number of sera identified by the reference assays as negative (b+d), expressed as a percentage. Specificity = d b + d Confidence intervals The 95% confidence intervals were calculated for values in order to assess the level of uncertainty introduced by sample size, etc. Exact 95% confidence intervals for binomial proportions were calculated from the F-distribution. (Armitage, 2002; Kirkwood, 2003] Predictive values The positive predictive value is the probability that when the test is reactive that the specimen does contain antibody to HIV. PPVs were calculated using the formula. PPV = ( prevalence)( sensitivity) ( prevalence)( sensitivity) + ( 1 prevalence)( 1 specificity) The negative predictive value is the probability that when the test is negative that a specimen does not have antibody to HIV. NPVs were calculated using the formula. NPV = ( 1 prevalence)( specificity) ( 1 prevalence)( specificity) + ( prevalence)( 1 sensitivity) The probability that a test result 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 PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 10 of 27

11 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 individuals testing falsepositive decreases; conversely, the likelihood that a person whose test result is negative is truly uninfected (i.e., the negative predictive value [NPV]), decreases as prevalence increases. Therefore, as prevalence increases, so does the proportion of individuals testing falsenegative. The PPV and NPV are calculated at a prevalence of 0.1%, 1% and 5% Indeterminate results For the WHO specimen reference panel only: specimens which are found to be indeterminate by the criteria stated in the instructions for use were retested in duplicate on the same batch number of assay. In the case that the testing result could not be resolved after all testing, the specimen was called indeterminate and included in sensitivity/specificity calculations. Values for initial sensitivity and specificity were calculated based on the results obtained for the assay under evaluation on the first batch. The final sensitivity and specificity values were calculated taking into consideration the repeat testing performed on a same batch and further testing second batch of the assay under evaluation Discrepant results For the WHO specimen reference panel only: those specimens with results that were consistent with the reference testing results, underwent no further testing. Those specimens with results discrepant from the reference result were retested in duplicate using the same batch number by the same operator. The results that occurred two out of three times were recorded as the result for that particular batch. If the result was again discrepant, the specimen was repeated on the second batch, if available. If the result on the second batch was concordant with the reference testing results, no further testing was required. If the result was still discrepant from the reference results, the result was recorded as is. The values for initial sensitivity and specificity were calculated based on the first results obtained for HIV 1/2 STAT-PAK on the first batch. The final sensitivity and specificity were calculated taking into consideration the repeat testing performed on a same batch and further testing on the second batch. The results of any discrepant results are presented in Annex Interpretation of results from batch-to-batch panel The results of the batch-to-batch variation panel on the two batches by HIV 1/2 STAT-PAK was compared and a variation of +/- 1 dilution series was considered acceptable. The results are presented in Annex Interpretation of results from HIV seroconversion panels The results obtained with seroconversion panels from individuals in the early stages of HIV infection from HIV 1/2 STAT-PAK were compared with those results obtained using Enzygnost Anti-HIV 1/2 Plus (Siemens Healthcare Diagnostics), the assay arbitrarily designated the reference for determination of relative sensitivity in these panels. For each seroconversion panel, the first specimen in the sample sequence to become reactive with Enzygnost Anti-HIV 1/2 Plus was assigned the value 0. Results from HIV 1/2 STAT-PAK were compared with Enzygnost Anti-HIV 1/2 Plus 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 the assay under evaluation. For example, if the assay became reactive two specimens earlier in a panel than Enzygnost Anti-HIV 1/2 Plus, the PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 11 of 27

12 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, the value assigned was +1. The assigned values over the eight seroconversion panels were averaged to determine a mean relative seroconversion sensitivity index for HIV 1/2 STAT-PAK and the 95% confidence intervals were determined. All specimens were also tested on the Vironostika HIV Ag/Ab (biomérieux), INNOTEST HIV Antigen mab (Innogenetics) and INNO-LIA HIV I/II Score line immunoassasy (Innogenetics). The results are for the seroconversion panels are presented in Annex Interpretation of results from HIV mixed titer panel The number of specimens detected by HIV 1/2 STAT-PAK on the HIV mixed titer performance panel was determined by comparison with the anti-hiv status assigned (expected results) following interpretation of the combined reference testing results generated by the following assays: Enzgnost Anti-HIV 1/2 Plus EIA (Siemens Healthcare Diagnostics), Vironostika HIV Ag/Ab (biomérieux), INNO-LIA HIV I/II Score line immunoassay (Innogenetics), and INNOTEST HIV Antigen mab (Innogenetics). The results are presented in Annex Interpretation of results from WHO reference preparations The number of specimens detected by HIV 1/2 STAT-PAK for the WHO biological reference preparation panel (1 st International Reference Panel for anti-hiv [NIBSC code 02/210]) was determined by comparison with the interpretation of the combined reference testing results generated by the following assays: Enzgnost Anti-HIV 1/2 Plus EIA (Siemens Healthcare Diagnostics), Vironostika HIV Ag/Ab (biomérieux), INNO-LIA HIV I/II Score line immunoassay (Innogenetics), and INNOTEST HIV Antigen mab (Innogenetics). The results are presented in Annex Inter-reader variability Three individuals independently interpreted each test result. The inter-reader variability was expressed as the percentage of specimens for which initial test results were differently interpreted (i.e. positive, negative, indeterminate) by the independent readers. The results are presented in Annex 8. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 12 of 27

13 6. Results 6.1. Validation of results All test runs were valid based on the run validation criteria outlined in the instructions for use. The control band was visible on all devices Comparison of initial testing results with reference results Detection of antibodies to HIV-1/2 Reference results + - HIV 1/2 STAT-PAK initial results Sensitivity (95% CI): 418/421 = 99.29% (97.9%-100.0%) Specificity (95% CI): 658/658 = 100% (99.4%-100.0%) Indeterminate results: 0/1079 = 0% False positive results: 0/1079 = 0% (see Annex 3) False negative results: 3/1079 = 0.3% (see Annex 3) Comparison of final testing results with reference results Detection of antibodies to HIV-1/2 Reference results + - HIV 1/2 STAT-PAK final results Sensitivity (95% CI): 419/421 = 99.52% (98.3%-99.9%) Specificity (95% CI): 658/658 = 100.0% (99.4%-100.0%) Indeterminate results: 0/1079 = 0% False positive results: 0/1079 = 0% (see Annex 3) False negative results: 2/1079 = 0.2% (see Annex 3) Discrepant results False non-reactive results Upon initial testing on batch , three specimens (WHO3-067, WHO and WHO ) returned a false non-reactive test result. Two of the specimens (WHO and WHO ) remained false non-reactive after repeat testing in duplicate using the same batch. Both specimens remained false non-reactive when tested with the other batch The results are presented in Annex 3. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 13 of 27

14 6.5. Predictive values PPV (0.1% prevalence) : 100% PPV (1% prevalence) : 100% PPV (5% prevalence) : 100% NPV (0.1% prevalence) : 99.99% NPV (1% prevalence) : 99.99% NPV (5% prevalence) : 99.98% 6.6. Results from batch-to-batch panel Batch A ( ) of HIV 1/2 STAT-PAK detected 47/160 members of the dilution panel. Batch B ( ) of HIV 1/2 STAT-PAK detected 47/160 members of the dilution panel. The results are presented in Annex Results from HIV seroconversion panels The HIV 1/2 STAT-PAK detected antibodies to HIV in 8 different seroconversion panels, on average, more than specimens later than the reference assay (Enzygnost Anti-HIV 1/2 Plus [Siemens Healthcare Diagnostics]). The results are for the seroconversion panels are presented in Annex Results from HIV mixed titer panel The HIV 1/2 STAT-PAK detected 19/25 specimens in the HIV mixed titer panel. The results are presented in Annex Results from WHO reference preparations The HIV 1/2 STAT-PAK detected 6/6 specimens in the WHO reference panel. The results are presented in Annex Inter-reader variability Given the test is of simple/rapid format, the inter-reader variability was calculated and found to be 0.18%. The results are presented in Annex 8. PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 14 of 27

15 7. Appraisal by laboratory technician Rating* Clarity Kit instructions: Presentation Content Safety instructions Kit/reagent packaging and labelling: Clear Labelling Safety Specimen type Serum/plasma used Specimen dispensing and volume: Specimen volume 5 µl Reagent dispensing: Equipment required: Specimen Yes addition control Reagent Yes addition control Equipment Yes required Details of Timer equipment required Number of steps to completion of test: Number 2 Endpoint stability: Time 5 mins Time from start to completion: Time 17 mins Recommended maximum tests per Test maximum 10 run: Actual number of tests possible per run: Test number 10 Other comments: The use of serum/plasma specimens make it more difficult to verify that the opening of the loop is filled with specimen. * Rating key: : 1 = poor; 2 = needs improvement; 3 = satisfactory; 4 = good; 5 = excellent PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 15 of 27

16 8. Summary The HIV 1/2 STAT-PAK (CHEMBIO Diagnostics Systems, Inc) was evaluated by WHO in the fourth quarter of using serum/plasma specimens. From this evaluation, we drew the following conclusions: The HIV 1/2 STAT-PAK (CHEMBIO Diagnostics Systems, Inc) is an immunochromatographic assay for the detection of antibodies to HIV in human serum/plasma, and fingerstick/venous whole blood specimens. A volume of 5 µl of specimen is needed to perform the assay. This type of assay requires no sophisticated equipment and can therefore be performed in laboratories with limited facilities. Reading of the results can be done visually. In this limited evaluation on a panel of 1079 specimens, we found an initial sensitivity (95% CI of 99.29% (97.9% %) and an initial specificity (95% CI) of 100% (99.4% - 100%) compared to the reference assays. The final sensitivity (95% CI) was 99.52% (98.3% - 100%) and the final specificity (95% CI) was 100% (99.4% - 100%) compared to the reference assays. In this study, 0% of the results were recorded as indeterminate. Results were interpreted independently by three technicians; the inter-reader variability was 0.18%. The invalid rate was 0%. The detailed data of this evaluation will be published in an official WHO report of the operational characteristics of commercially available assays to detect antibodies to HIV in human serum/plasma. The report will be made available on the WHO web site at: PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 16 of 27

17 9. References Armitage P, Berry G, Matthews J. Statistical Methods in Medical Research, 4 th ed. Oxford, Blackwell Science Ltd, 2002 Kirkwood B, Sterne J. Essential Medical Statistics, 2 nd ed. Oxford, Blackwell Science Ltd, 2003 PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 17 of 27

18 10. Annex 1 Instructions for use for HIV 1/2 STAT-PAK PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 18 of 27

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30 Annex 1: Results of HIV Positive Specimens Results Legend 0 = Negative RA = Result of Reader A R= Reactive HIV 1/2 STAT-PAK 1 = Uncertain reactivity, indeterminate RB = Result of Reader B NR = Non-reactive Test 1 HIV 1/2 STAT-PAK Results Vironostika Vironostika Enzygnost Result Test 2 Result Test 3 Result Final HIV HIV Anti-HIV Reference Results INNO-LIA HIV I/II Score LIA Result Uniform II Ag/Ab 1/2 Plus REFERENCE WHO Batch 1 Batch 2 Batch 3 plus O Result RESULT Number Origin No RA RB RC No RA RB RC No RA RB RC OD/CO OD/CO OD/CO sgp120 gp41 p31 p24 p17 sgp105 gp36 WHO2-714 Africa A R R >=6, HIV-2 Positive WHO2-719 Africa A R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-720 Africa A R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-722 Africa A R R >=6, HIV-2 Positive WHO2-723 Africa A R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-724 Africa A R R >=15,08 >19.6 >=6, HIV Positive WHO2-725 Africa A R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-726 Africa A R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-727 Africa A R R >=15,08 >19.6 >=6, HIV Positive WHO2-728 Africa A R R >=15,08 >19.6 >=6, HIV Positive WHO2-730 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-733 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-734 Africa B R R >=15,08 >19.6 >=6, HIV Positive WHO2-735 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-737 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-738 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-739 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-740 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-741 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-742 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO2-754 Africa B R R >=15,08 >19.6 >=6, HIV-2 Positive WHO L America A R R >=19,4 >18.6 >=6, HIV Positive WHO L America A R R 14.0 >18.6 >=6, /- - - HIV-1 Positive WHO L America A R R >=19,4 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R 14.8 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R 15.4 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R 16.0 >18.6 >=6, HIV-1 Positive WHO L America A R R 16.4 >18.6 >=6, HIV Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO L America A R R >=18,9 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Europe A R R 16.6 >18.6 >=6, HIV-1 Positive PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 1 of 10

31 Annex 1: Results of HIV Positive Specimens Results Legend 0 = Negative RA = Result of Reader A R= Reactive HIV 1/2 STAT-PAK 1 = Uncertain reactivity, indeterminate RB = Result of Reader B NR = Non-reactive Test 1 HIV 1/2 STAT-PAK Results Vironostika Vironostika Enzygnost Result Test 2 Result Test 3 Result Final HIV HIV Anti-HIV Reference Results INNO-LIA HIV I/II Score LIA Result Uniform II Ag/Ab 1/2 Plus REFERENCE WHO Batch 1 Batch 2 Batch 3 plus O Result RESULT Number Origin No RA RB RC No RA RB RC No RA RB RC OD/CO OD/CO OD/CO sgp120 gp41 p31 p24 p17 sgp105 gp36 WHO Europe A R R 14.4 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Africa A R R 17.9 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Europe A NR A R A R R >=20,3 >18.6 >=6, / HIV-1 Positive B NR B R B R R WHO Europe A R R 16.3 >18.6 >=6, HIV-1 Positive WHO Africa A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Africa A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=6, HIV-1 Positive WHO Europe A R R >=20,3 >18.6 >=6, HIV-1 Positive WHO Europe A R R 15.4 >18.6 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >20.7 >=6, HIV-1 Positive WHO Europe A R R 13.0 >20.7 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >20.7 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >20.7 >=6, HIV-1 Positive WHO Europe A R R >=20,3 >20.7 >=6, / HIV-1 Positive WHO Europe A R R 17.5 >20.7 >=6, HIV-1 Positive WHO Europe B R R 15.8 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=20,3 >20.7 >=6, HIV-1 Positive WHO Africa B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R 13.1 >20.7 >=6, HIV-1 Positive WHO Europe B R R 16.9 >20.7 >=6, HIV-1 Positive WHO Africa B R R 17.3 >20.7 >=6, HIV-1 Positive WHO Africa B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Africa B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R 17.1 >20.7 >=6, HIV-1 Positive WHO Europe B R R 15.0 >20.7 >=6, HIV-1 Positive WHO L America B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Africa B R R >=6, HIV-1 Positive WHO Europe B R R >=6, HIV-1 Positive WHO Africa B R R 14.9 >20.7 >=6, HIV-1 Positive WHO Africa B R R >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO Europe B R R >=19, >=6, HIV-1 Positive WHO Africa B R R >=6, HIV-1 Positive WHO Africa B R R >=19, >=6, HIV-1 Positive WHO Europe B R R >=19,0 >20.7 >=6, HIV-1 Positive WHO L America B R R 13.0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 2 of 10

32 Annex 1: Results of HIV Positive Specimens Results Legend 0 = Negative RA = Result of Reader A R= Reactive HIV 1/2 STAT-PAK 1 = Uncertain reactivity, indeterminate RB = Result of Reader B NR = Non-reactive Test 1 HIV 1/2 STAT-PAK Results Vironostika Vironostika Enzygnost Result Test 2 Result Test 3 Result Final HIV HIV Anti-HIV Reference Results INNO-LIA HIV I/II Score LIA Result Uniform II Ag/Ab 1/2 Plus REFERENCE WHO Batch 1 Batch 2 Batch 3 plus O Result RESULT Number Origin No RA RB RC No RA RB RC No RA RB RC OD/CO OD/CO OD/CO sgp120 gp41 p31 p24 p17 sgp105 gp36 WHO Europe B R R 12.1 >19.2 >=6, /- - - HIV-1 Positive WHO Europe B R R 14.6 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,0 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,4 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,4 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe B R R 16.9 >19.2 >=6, HIV-1 Positive WHO Africa B R R 17.7 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >19.2 >=6, HIV-1 Positive WHO L America A R R >=19,5 >19.2 >=6, / HIV-1 Positive WHO Europe A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe A R R 13.9 >19.2 >=6, HIV-1 Positive WHO Europe A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Africa A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe A R R 17.5 >19.2 >=6, HIV-1 Positive WHO Europe A R R >=19,5 >19.2 >=6, HIV-1 Positive WHO Europe A R R 12.1 >19.2 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,5 >24.8 >=6, HIV-1 Positive WHO Africa B R R >=19,6 >24.8 >=6, HIV-1 Positive WHO Africa B R R >=19,6 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,6 >24.8 >=6, HIV-1 Positive WHO Europe B R R 15.7 >24.8 >=6, HIV-1 Positive WHO Europe B R R 10.5 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,6 >24.8 >=6, HIV-1 Positive WHO Europe B R R >=19,6 >24.8 >=6, / HIV-1 Positive WHO Europe B R R >=19,6 >24.8 >=6, HIV-1 Positive WHO Africa B R R 17.0 >24.8 >=6, HIV-1 Positive WHO Africa B R R 15.7 >24.8 >=6, HIV-1 Positive WHO Europe B R R 14.1 >24.8 >=6, HIV-1 Positive WHO Europe B R R 16.1 >24.8 >=6, HIV-1 Positive WHO Europe A R R >=19, >=6, HIV-1 Positive WHO Europe A R R >=6, HIV-1 Positive WHO Europe A R R >=6, HIV Positive WHO Europe A R R >=6, HIV-1 Positive WHO Africa A R R >=6, HIV-1 Positive PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 3 of 10

33 Annex 1: Results of HIV Positive Specimens Results Legend 0 = Negative RA = Result of Reader A R= Reactive HIV 1/2 STAT-PAK 1 = Uncertain reactivity, indeterminate RB = Result of Reader B NR = Non-reactive Test 1 HIV 1/2 STAT-PAK Results Vironostika Vironostika Enzygnost Result Test 2 Result Test 3 Result Final HIV HIV Anti-HIV Reference Results INNO-LIA HIV I/II Score LIA Result Uniform II Ag/Ab 1/2 Plus REFERENCE WHO Batch 1 Batch 2 Batch 3 plus O Result RESULT Number Origin No RA RB RC No RA RB RC No RA RB RC OD/CO OD/CO OD/CO sgp120 gp41 p31 p24 p17 sgp105 gp36 WHO Europe A R R >=6, HIV-1 Positive WHO Europe A R R >=19,6 6.8 >=6, HIV-1 Positive WHO Europe A R R >=6, HIV-1 Positive WHO Africa A R R >=19,6 7.1 >=6, HIV-1 Positive WHO Europe A R R >=19,6 >19.4 >=6, HIV-1 Positive WHO Europe A R R 14.1 >19.4 >=6, HIV-1 Positive WHO Europe A R R 15.2 >19.4 >=6, HIV-1 Positive WHO Africa A R R 16.0 >19.4 >=6, HIV-1 Positive WHO Africa A R R 12.5 >19.4 >=6, HIV-1 Positive WHO Europe A R R >=19,6 >19.4 >=6, / HIV-1 Positive WHO Europe A R R >=19,6 >19.4 >=6, HIV-1 Positive WHO Africa B R R 15.7 >19.4 >=6, HIV-1 Positive WHO Europe B R R >=19,4 >19.4 >=6, HIV-1 Positive WHO Europe B R R 12.5 >22.6 >=6, HIV-1 Positive WHO Africa B R R >=19, >=6, HIV-1 Positive WHO Europe B R R 17.4 >22.6 >=6, HIV-1 Positive WHO Europe B R R >=19,4 >22.6 >=6, /- 1+ +/- - - HIV-1 Positive WHO Europe B R R >=19,4 >22.6 >=6, HIV-1 Positive WHO Europe B R R >=19,4 >22.6 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R HIV-1 Positive WHO L America B R R 13.5 > HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America B R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R 14.6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R 12.3 >18.9 >=6, HIV-1 Positive WHO L America A R R 14.9 >18.9 >=6, HIV Positive WHO L America A R R 14.2 > HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R 14.6 >18.9 >=6, HIV-1 Positive WHO L America A R R 13.0 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >18.9 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R 15.0 >19.6 >=6, HIV-1 Positive PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 4 of 10

34 Annex 1: Results of HIV Positive Specimens Results Legend 0 = Negative RA = Result of Reader A R= Reactive HIV 1/2 STAT-PAK 1 = Uncertain reactivity, indeterminate RB = Result of Reader B NR = Non-reactive Test 1 HIV 1/2 STAT-PAK Results Vironostika Vironostika Enzygnost Result Test 2 Result Test 3 Result Final HIV HIV Anti-HIV Reference Results INNO-LIA HIV I/II Score LIA Result Uniform II Ag/Ab 1/2 Plus REFERENCE WHO Batch 1 Batch 2 Batch 3 plus O Result RESULT Number Origin No RA RB RC No RA RB RC No RA RB RC OD/CO OD/CO OD/CO sgp120 gp41 p31 p24 p17 sgp105 gp36 WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R 14.2 >19.6 >=6, HIV-1 Positive WHO L America A R R 14.6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R 13.8 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R 13.5 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >19.6 >=6, HIV-1 Positive WHO L America A R R 14.0 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 11.9 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 14.3 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 10.0 >16.6 >=6, HIV-1 Positive WHO L America A R R 13.5 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 10.8 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 13.9 >16.6 >=6, HIV-1 Positive WHO L America A R R 14.6 >16.6 >=6, HIV-1 Positive WHO L America A R R 10.9 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 13.6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive WHO L America A R R 13.5 > HIV-1 Positive WHO L America A R R 14.2 >16.6 >=6, HIV-1 Positive WHO L America A R R >=16,6 >16.6 >=6, HIV-1 Positive PQDx_ HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) Page 5 of 10