|Year : 2012 | Volume
| Issue : 4 | Page : 397-402
Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India
HS Iqbal, S Solomon, KG Murugavel, SS Solomon, P Balakrishnan
Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), Voluntary Health Services Hospital Campus, Taramani, Chennai, India
|Date of Submission||05-Mar-2012|
|Date of Acceptance||22-May-2012|
|Date of Web Publication||24-Nov-2012|
H S Iqbal
Infectious Diseases Laboratory, YRG Centre for AIDS Research and Education (YRG CARE), Voluntary Health Services Hospital Campus, Taramani, Chennai
Source of Support: None, Conflict of Interest: None
Purpose: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. Materials and Methods: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. Results: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. Conclusions: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.
Keywords: ELISA, HIV, kit evaluation, western blot, p24 Ag
|How to cite this article:|
Iqbal H S, Solomon S, Murugavel K G, Solomon S S, Balakrishnan P. Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India. Indian J Med Microbiol 2012;30:397-402
|How to cite this URL:|
Iqbal H S, Solomon S, Murugavel K G, Solomon S S, Balakrishnan P. Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India. Indian J Med Microbiol [serial online] 2012 [cited 2020 Aug 8];30:397-402. Available from: http://www.ijmm.org/text.asp?2012/30/4/397/103758
| ~ Introduction|| |
Infection with human immunodeficiency virus (HIV), which causes AIDS, has become a worldwide epidemic and become one of the major public health concerns for all countries. ,, Early diagnosis of HIV infection is an important strategy for prevention and patient management. ,, Although several different assays are presently available for the detection of specific antibodies to diagnose HIV infection, evaluation reports of these commercially available assays are very limited.
HIV is diagnosed mainly by using rapid and enzyme-linked immunosorbent assays (ELISA) and the gold standard assays like western blot/immunofluorescence/radio immuno precipitation tests are only used for resolving discrepancies in resource-limited settings. ,, Both ELISA and rapid diagnostic assays have got their own merits and demerits in the diagnosis of HIV infection. Conventional ELISA testing for HIV is an efficient way to screen large numbers of specimens at one time  with reduced cost. The inclusion of anti-p24 antibodies in the solid phase ELISA have enabled the diagnosis of p24 antigen to reduce the window period in fourth generation assays. However, the disadvantages of the ELISA are the need for well-trained technical manpower, appropriate expensive equipment and delay due to batch testing.  Moreover, with rapid assays it would be possible to give results for individual specimens with the sensitivity and specificity equal to ELISA assays. In recent years, few manufacturers have come out with rapid assays stable at room temperature (RT) or up to 30°C. But these rapid assays are relatively costlier when compared with ELISA tests.
The national HIV testing algorithm was devised by Government of India,  which emphasizes the need for at least three different HIV diagnostic assays (using different antigenic principles and/or different methodologies) to be applied in diagnostic settings mainly for voluntary counselling and testing centre (VCTC) clients. At the same time increased performance characteristics of the available diagnostic assays are needed to get best out of these algorithms. In the present study we report the performance characteristics of four different indigenous commercially available rapid and ELISA assays for the diagnosis of HIV infection.
| ~ Materials and Methods|| |
This study was carried out at a one of the largest tertiary HIV referral centre in Chennai, India. It has 65% of the HIV prevalence among VCT clients. In the present evaluation, the following two batch of specimens (n=180; n=20) were subjected to HIV testing by (i) Comb AIDS - RS Advantage HIV 1+2 Immunodot test; (ii) Enzaids HIV 1+2 ELISA test; (iii) Enzaids Duet HIV Antigen+antibody ELISA test; (iv) Signal HIV Flow Through HIV 1+2 Test kits. All these products were manufactured by Span Diagnostics Ltd, Surat, India. A double blinding technique was adopted to maintain the patient confidentiality. The first person obtained the specimens and generated duplicate identification numbers and the second person performed the test; results were analyzed by both of them.
A batch of 180 specimens with the following characteristics were included. Of them, 90 specimens were confirmed positive for HIV-1 by HIV western blot assay (Genetic systems TM HIV-1 western blot, Bio-rad Laboratories, Redmond, WA, USA) and rest (n=90) of the specimens were confirmed HIV negative by double ELISA method, that is, one fourth generation (Vironostika HIV Uni-Form II Ag/Ab, Biomerieux, Boxtel, the Netherlands) and another third generation (HIV-1.2.0 Murex Biotech Limited, Dartford, UK) ELISA assays were used for the confirmation. There are another batch of 20 specimens that were also used for the evaluation. These are selected specimens from our VCT cohort with HIV indeterminate results in our HIV testing algorithm, some of these specimens are from (i) individuals with discrepant results in different assays. (ii) Some clients having discordant results in different clinics are also referred here. These specimens were previously tested on the following kits (i) First response HIV card test 1-2.0 (PMC Medical India Pvt, Ltd, Daman, India), (ii) Determine HIV 1/2 (Inverness Medical Japan Co. Ltd, Tokyo, Japan), (iii) HIV TRI-DOT (BIOMED Industries, Parwanoo, India) assays. In addition, these specimens (n=20) were subjected to QualicodeTM HIV-1/2 (Immunetics Inc, Boston, MA, USA) and western blot assay for the confirmation of HIV infection. The HIV-1 infection was considered positive in the presence of any two or more of the following bands p24, gp41, gp120/160. Furthermore, the presence of any band not matching the positive criteria is considered as HIV-1 indeterminate. There was also one HIV-2 envelope band present in the strip to rule out HIV-2 infection.
P24 Ag detection: The sensitivity of "Enzaids Duet HIV Antigen+antibody fourth generation ELISA assay" for detecting p24 Ag was performed by standard dilution methods. A recombinant p24 (1 mg/mL), 'Sudarshan Biotech Limited', Hyderabad, India was used. From the source concentration 10 μg/mL p24 Ag concentration was prepared by diluting 1:100 with HIV negative human serum, and 1 ng/mL solution was prepared by repeating 1:100 dilution additionally for two more times. Again 100 pg/mL p24 Ag solution was prepared by making 1:10 dilution with available p24 Ag dilution (1 ng/mL), from this point forward a doubling dilution was made to get the concentration on of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 pg/mL. These concentrations of p24 solution was treated as individual specimens and tested on EnzAids Duet HIV assay to find out the minimum concentration giving positive results.
The performance characteristics were determined for different assays using the 180 specimens from first batch and 8 (specimens confirmed to be positive or negative by qualicode western blot assay) from second batch of specimens. The following formulas were used to calculate test indices:  sensitivity=[TP/(TP+FN)]×100; specificity=[TN/(TN+FP)]×100; positive predictive value (PPV)=[TP/(TP+FP)]×100; negative predictive value (NPV)=[TN/(TN+FN)]×100; efficiency=[(TP+TN)/(TP+FN+TN+FP)] ×100; (where TP=number of true positives, TN=number of true negatives, FN=number of false negatives and FP=number of false positives).
| ~ Results|| |
The results with characterized specimens are shown in [Table 1]. All the HIV-1 western blot confirmed positive specimens are correctly identified by all the assays. The performance characteristics of three assays (CombAIDS, Enzaids HIV 1+2 and Enzaids Duet HIV) were comparable with gold standard assays results. The sensitivity, specificity, PPV, NPV and efficiency of these assays are found to be 100%. However, there was one HIV negative specimen that was identified as positive by Signal HIV assay reducing its specificity, PPV and efficiency. The Enzaids Duet HIV kit was found to be extreme sensitivity in detecting p24 Ag with the sensitivity of 1.5 pg/mL.
|Table 1: Performance characteristic of four HIV antibody tests used for comparative evaluation|
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The second batch of characterized specimens (n=20) were analyzed separately. The detailed individual specimen results are shown in [Table 2]. We found six specimens that were confirmed positive for HIV-1 by western blot assay of which three were found to be co-infected with HIV-2. All these specimens were found to be HIV positive with all the assays, which were evaluated. The HIV-2 infected specimens were correctly identified with First Response and TRIDOT assays. However, TRIDOT was unable to pickup five HIV-1 western blot positive specimens. Of the total 20 specimens, 9 specimens were found to be HIV-1 indeterminate with western blot bands not matching positive criteria, many of these indeterminate specimens were found to be positive with rapid assays. However, few HIV-1 indeterminate specimens are not being picked up by some rapid assays. Three of these were found to be HIV-2 positive in western blot, and were also positive for HIV-2 with the kits, which were able to differentiate between HIV-1 and HIV-2. There are two specimens (s# 19 and 20) found to be positive for HIV in Inverness Determine and First Response assay, which were confirmed to be negative for HIV-1 and HIV-2 antibodies by western blot assay.
|Table 2: Individual specimens comparison between different HIV rapid/ELISA assays and HIV western blot|
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| ~ Discussion|| |
In the present study we have evaluated the performance of two indigenous rapid and two ELISA kits. The Comb AIDS kit was able to pickup HIV-1 indeterminate specimens better than other kits under evaluation. The First response and Determine HIV 1/2 assays have picked up all the HIV-1 indeterminate specimens but it should also be kept in mind that these kits have shown false positive for two specimens, which were actually negative for HIV-1 and HIV-2 in the western blot assay. Government of India  recommends a minimum of three HIV kits to be used in VCT centres. It can be the combination of rapid and/or ELISA assays using different antigenic principles and/or methodologies. The western blot assay is no longer used for the confirmation of HIV infection in the diagnostic settings. Additionally, the combination of two or more antibody screening assays, with different antigenic bases, will be more specific and sensitive than western blot. ,, Hence evaluating the diagnostic kits always gain importance in selecting the commercially available assays. As per the current evaluation all the kits can be used in the HIV testing algorithm.
It will be a hard time for the VCT clients to get their HIV indeterminate results. Usually they will be asked to come after 15 days. It will be an unnecessarily mental anxiety, psychological stress to the patient if the indeterminate results are due to false positive result in any of the assays used in the testing algorithm. It can also incur additional expenses to the VCT clients on travel, loss of wages and testing, as individuals from rural areas need to travel hundreds of miles to access HIV testing facilities in government or private sectors. The false negative results must also be given importance because (i) if the less sensitive assay is used as the first test in serial testing algorithm then it can be responsible for silent HIV transmissions in the community and the (ii) threat of transfusing falsely diagnosed (false negative) HIV positive blood units.
The fourth generation ELISA assays gain its own importance in detecting p24 Ag. In the current evaluation the Enzaids Duet has shown extreme sensitive in detecting the p24 Ag it is much higher than the detecting limits of the other p24 detecting assays like 'HIV-1 Capsid protein p24 ELISA pair set',  Vironostika HIV-1 p24 antigen assay',  'AxSym', 'Murex' and 'Vidas HIV Duo Ultra'  assays. p24 detection assays are helpful in diagnosing HIV infection in much earlier time periods from the day of infection, thereby giving opportunity to the counsellor to sensitize the patient and stop spreading infection. At the same time care must be taken while getting discrepant result between third and fourth generation assays; the assay must be repeated after a fortnight. In our evaluation we did not see positive result in Enzaids Duet assay for some of these specimens for which we observed p24 band in western blot test. This could be due to false indeterminate in western blot, ,, further tests are required to resolve the conflicting serology of these specimens.
There are little previous studies with the kits under evaluation, however, Enzaids and Comb AIDS assays have shown good performance characteristics in concordant with their previous evaluations. , Enzaids has shown best performance characteristics when used in single, as well as series with other kits in one of the recent evaluations from the country.  There are some reports available on the performance of the other kits used in this study. The TRIDOT kit has not detected few HIV-1 western blot positive specimens and has falsely detected one specimen as HIV-2 in concordant with its previous reports. , Moreover, the Determine HIV 1/2 assay has shown good performance characteristics, which is also seen in other Indian  and international reports. ,
Even though all of these assays have shown excellent performance characteristics the differentiation of HIV-1 and HIV-2 has got its own social importance. The differentiation is required for monitoring and disease management purpose. Hence in any given HIV testing algorithm it is always better to use at least two kits, which has the capacity to differentiate between HIV-1 and HIV-2. Further evaluations are needed to check the performance characteristics of kits, which have the capacity to differentiate between HIV-1 and HIV-2. Moreover, the available national algorithms  are not clear on the differentiation between HIV-1 and HIV-2.
Having discussed all these, the essence of the manuscript is in resolving conflicting serology of discrepant HIV results, especially in HIV care and referral centres. Discrepancy specimens can be from other laboratories or from the same laboratory, the user has to ensure the first assay used in serial testing algorithm must be highly sensitive and the following must be highly specific. For the discrepant results (unable to declare as positive due to negative in any one assay in a given algorithm) it is recommended to repeat the test with new specimen rather than going for western blot. While western blot again can give indeterminate results with incomplete antibody production and in conditions [haematological malignancies, autoimmune disorders (Eg, Systemic lupus erythematosus SLE), primary biliary cirrhosis, HIV vaccine trial participants, Stevens-Johnson syndrome More Details and anti- Human human leukocyte (HLA) antibody etc.] for indeterminate results. With kits having potential to differentiate between HIV-1 and HIV-2 there is a possibility of getting cross reactions of HIV-2 with HIV-1 specimen and vice versa. In our experience HIV TRIDOT kit works fine for HIV-2 detection and first response for HIV-1 status. HIV serology for infants at 18 months must be repeated with caution because there may be existing maternal HIV residual antibodies even at 18 months for the infants. In our centre HIV antibody positive results (at 18 months; data not shown) were obtained for infants with repeated DNA PCR negative results (Before attaining 1 year). Hence one should keep these in mind while interpreting HIV results.
Even though this study has evaluated the performance of different indigenous assays with western blot results, it has the following limitation. We have used neither HIV- 2 western blot assay to confirm HIV-2 infection nor have we done any molecular assays for the differentiation and confirmation of HIV status. We have not done the p24 Ag ELISA to find out the actual status of HIV-1 indeterminate specimens with p24 bands. The result from follow-up specimens is also not available. Our evaluation did not include any proficiency panel specimens from regulatory bodies and we have not used any Boston Biomedical Inc, (BBI) or seroconversion panels. Moreover, in our evaluation we have not got the patient's clinical characteristics such as (i) any auto immune disease, (ii) increased IgG levels and other (iii) viral infections which can interact with the results or the information on usage of any recreational drugs.
All these kits used in this evaluation are indigenous, locally available, low cost, widely distributed in the country and affordable even for small laboratories with best performance characteristics equal to imported kits. ,, These kits can be used in point of care facilities, emergency rooms, anti-natal clinics, reference laboratories also in places with limited infra structure. Hence to conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. The HIV testing assays Comb AIDS - RS Advantage HIV 1+2 Immunodot Test; (ii) Enzaids HIV 1+2 ELISA test; (iii) Enzaids Duet HIV Antigen+antibody ELISA test; (iv) Signal HIV Flow Through HIV 1+2 tests have got excellent performance characteristics and much suitable for in use for resource-limited settings.
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[Table 1], [Table 2]
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