Open Access

Comparative assessment of commercial ELISA kits for detection of HIV in India

  • Srijita Nandi1,
  • Susmita Maity1,
  • Somesh Chandra Bhunia1 and
  • Malay Kumar Saha1Email author
BMC Research Notes20147:436

https://doi.org/10.1186/1756-0500-7-436

Received: 31 July 2013

Accepted: 1 July 2014

Published: 7 July 2014

Abstract

Background

India harbors the 3rd highest HIV infected population globally. The magnitude of the HIV detection challenge is enormous. ELISA is the most commonly used screening technique for HIV. There is always an acute need for good quality ELISA kits. However, the quality evaluation data on Indian kits are very limited in comparison with internationally recognized kits. This study aimed to evaluate the performance and diagnostic usefulness of five commercially available ELISA kits which are frequently used in India.

Findings

The ELISA kits evaluated using an in-house well characterized 100 member sera panel revealed 100% sensitivity for all the batches. However, batch to batch variation in terms of specificity, positive predictive value (PPV) and efficiency, although not statistically significant (p > 0.05), was observed. For specificity, the 3rd generation kits (mean 99.6% to 99.3%) were comparatively better than the 4th generation assays (97.2% to 96.9%). But the 4th generation kits performed far better in the ability for early detection post HIV infection in the 25 member commercial seroconversion panel with a margin of at least 22 days and as high as 35 days than the 3rd generation assays.

Conclusions

The commercial ELISA kits with 100% sensitivity seem appropriate for HIV screening. The ability of early detection post HIV infection favors use of 4th generation kits for ensuring HIV free blood for transfusion. Lot to lot variations, especially kits having the specificity level ≤98.0%, indicate the need for a regular mechanism of kit evaluation for each batch for procuring kits appropriate for intended use.

Keywords

HIV ELISA Sensitivity Specificity Efficiency Sera panel Seroconversion panel

Findings

Background

HIV is a major global public health issue [1]. For assuring a safe blood supply and preventing HIV infection, proper and accurate detection of HIV is essential [2]. In India, diagnosis of HIV infection is a major challenge [3, 4]. Several commercial assays are available for detection of HIV infection. ELISA is the most commonly used screening assay for HIV [2, 5]. A number of ELISA kits for HIV detection with different principles are available. Nowadays, in India 3rd generation ELISA are most commonly used. The 4th generation assays are based on combined detection of antigen and antibodies simultaneously and reduce the diagnostic window period further, compared to third generation ELISA which is based on anti-HIV antibody assay [68]. The improved sensitivity for ELISA is mostly accompanied by a decreased specificity. In an Indian perspective, limited articles on evaluation and performance of ELISA kits are available [9] though HIV testing is being done for a vast numbers of individuals as well as large number of specimens for ensuring HIV free safe blood for transfusion. Being the 2nd most populous country with the 3rd largest burden of HIV in the world [10], the magnitude of HIV testing challenge in India is enormous and the appropriate response to the challenge is to ensure the quality of the assay kits suitable for the intended use. This study aims to evaluate the quality of commonly available commercial ELISA kits for their ability to detect HIV suitable for appropriate use in India.

Materials and methods

The study was carried out at a National HIV Reference Laboratory designated for evaluation of diagnostic kits, including ELISA, in India. A well characterized, 100 members, in-house HIV serum panel was used to evaluate and compare the performance of the kits. The sera used for preparing the in-house panel were obtained anonymously from attendees of the Counseling and Testing Centre by taking informed consent as per the protocol approved by Institutional Ethical Committee of National Institute of Cholera and Enteric Diseases. Beside the negative and positive sera, this serum panel also contained low positive sera that have shown uniform results in all assays used for validation. The characterization of in-house panel was done using United States Food and Drug Administration (U.S. FDA) or Indian Central Drug Standard Control Organization (CDSCO) approved kits (Table 1). Samples non-reactive for all assays were defined as negative and reactive for all assays were defined as positive member in the panel. A commercial seroconversion panel (Lot# RP-018, Bio-Rad Laboratories, U.S.A) used to evaluate kits consists of a series of 25 specimens collected from an individual infected with HIV undergoing seroconversion.
Table 1

Details of kits used for characterization of in-house panel sera

Source of sample

Name of panel

Test details

  

ELISA 1

ELISA 2

Rapid test

Confirmatory test

ICTC

HIV Sera Panel (In-house)

Genetic Systems

Genedia HIV Ag-Ab ELISA

Rapid 1: Determine HIV1/2

Recombinant Immunoblot Assay

  

HIV-1/HIV-2

Green Cross Life Science Corp. Korea

Inverness Medical Japan Co. Ltd. Japan

Chiron RIBA HIV-1/HIV-2 SIA

  

Plus O EIA.

   
  

Bio-Rad Laboratories. USA

Reactivity range (pos specimens): 3.503-8.899

Rapid 2: HIV TRI-DOT + Ag

Ortho Clinical

     

Diagnostics Ltd, USA

    

J. Mitra & Co. Pvt. Ltd. India

AMPLICOR HIV-1 DNA Test

  

Reactivity range

   
     

Version 1.5

     

Roche Molecular Systems Inc. USA

  

(pos specimens): 3.485-10.017

   
Five batches each of 5 commonly available commercial ELISA kits, including 3rd and 4th generations, for HIV detection were evaluated (Table 2). All the kits were tested and results were validated strictly adhering to manufacturers’ instruction. The evaluation process maintained an unbiased method following a double blind procedure by using different personnel for pre-analytical and analytical testing sections. The final analysis of results with interpretation was done by the laboratory in-charge. The status of samples was unknown to the persons involved in pre-analytical and analytical procedures. The performance of kits was evaluated and compared in terms of 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) and efficiency ([(TP + TN)/(TP + FN + TN + FP)]×100), where TP = number of true positives, TN = number of true negatives, FP = number of false positives and FN = number of false negatives [11]. Confidence Interval (CI) was used to address precision of the proportion estimates and the degree of confidence was set to 95% [12]. Chi-square analysis was performed to assess the variation for specificity, PPV and efficiency among different kits as well as in different batches of same kit.
Table 2

Performance of HIV ELISA kits with In-house Sera Panel

Total no. of samples

Total no. of lots evaluated

Kit performance

100

5 Lots

J. Mitra & Co. Pvt. Ltd.

Span Diagnostics Ltd.

Transasia Bio-medicals Ltd.

Bio-Rad Laboratories.

Biomerieux.

(Confirmed Positive = 40 Confirmed Negative = 6 0)

for each kit

Assay result

Assay result

Assay result

Assay result

Assay result

   

Pos

Neg

Pos

Neg

Pos

Neg

Pos

Neg

Pos

Neg

 

Lot:1

Pos

40

00

40

01

40

00

40

02

40

02

Neg

00

60

00

59

00

60

00

58

00

58

Lot:2

Pos

40

01

40

00

40

00

40

02

40

02

Neg

00

59

00

60

00

60

00

58

00

58

Lot:3

Pos

40

00

40

00

40

00

40

01

40

01

Neg

00

0

00

60

00

60

00

59

00

59

Lot:4

Pos

40

01

40

00

40

00

40

01

40

03

Neg

00

59

00

60

00

60

00

59

00

57

Lot:5

Pos

40

00

40

01

40

01

40

02

40

01

Neg

00

60

00

59

00

59

00

58

00

59

Results

None of the ELISA kits evaluated was able to identify all the panel members correctly by all the batches (Table 2). But all the kits were found to be 100% sensitive in all the batches. Variation in batches of all the kits was evident in terms of specificity, PPV and efficiency (Table 3). ERBA LISA HIV 1 + 2 provided correct results in 4 batches by identifying all panel samples correctly with 99.8% efficiency. Microlisa HIV and Enzaids HIV 1 + 2 both performed equally by correct result only in 3 batches with 99.6% efficiency. The 4th generation kits, Genscreen and Vironostika, showed the false positivity rates higher than the 3rd generations, but the variations were not statistically significant in terms of specificity (χ 2 = 0.0683, df = 4, p > 0.05), PPV (χ 2 = 0.1253, df = 4, p > 0.05) and efficiency (χ 2 = 0.0230, df = 4, p > 0.05) of all batches of all the ELISA kits. The performance of the kits evaluated using seroconversion panel revealed that all the 3rd generation kits showed equal sensitivity by detecting HIV positivity. In contrast, the 4th generation kits, Genscreen (detected panel member 5, day 16) and Vironostika (detected member 7, day 29) were significantly more sensitive and were able to detect HIV positivity 35 and 22 days earlier respectively than the 3rd generation ELISA kits (Table 4).
Table 3

Performance characteristic of HIV ELISA kits used for comparative evaluation

Kit Name & Company

Lot

Kit Performance

  

Sensitivity

Specificity

PPV

NPV

Efficiency

  

(%)

%Mean (95% CI)

(%)

%Mean (95% CI)

(%)

%Mean (95% CI)

(%)

%Mean (95% CI)

(%)

%Mean (95% CI)

Microlisa HIV Company: J. Mitra & Co. Pvt. Ltd.

1

100

100

100

99.3

100

99.0

100

100

100

99.6

(100-100)

(98.2-100.3)

(97.5-100.4)

(100-100)

(98.9-100.2)

2

100

98.3

97.6

100

99.0

3

100

100

100

100

100

4

100

98.3

97.6

100

99.0

5

100

100

100

100

100

Enzaids HIV 1 + 2 ELISA Company: SPAN Diagnostics Ltd.

1

100

100

98.3

99.3

97.6

99.0

100

100

99.0

99.6

2

100

(100-100)

100

(98.2-100.3)

100

(97.5-100.4)

100

(100-100)

100

(98.9-100.2)

3

100

100

100

100

100

4

100

100

100

100

100

5

100

98.3

97.6

100

99.0

ERBA LISA HIV 1 + 2 Company: Transasia Bio-medicals Ltd.

1

100

100

100

99.6

100

99.5

100

100

100

99.8

2

100

(100-100)

100

(98.7-100.4)

100

(98.3-100.6)

100

(100-100)

100

(99.3-100.2)

3

100

100

100

100

100

4

100

100

100

100

100

5

100

98.3

97.6

100

99.0

Genscreen Plus HIV Ag-Ab ELISA Company: Bio-Rad Laboratories., U.S.A

1

100

100

96.6

97.2

95.4

96.2

100

100

98.0

98.4

2

100

(100-100)

96.6

(96.1-98.2)

95.4

(94.8-97.5)

100

(100-100)

98.0

(97.7-99.0)

3

100

98.3

97.6

100

99.0

4

100

98.3

97.6

100

99.0

5

100

96.6

95.4

100

98.0

Vironostika HIV Ag/Ab Company: Biomerieux SA. France

1

100

100

96.6

96.9

95.4

95.8

100

100

98.0

98.2

2

100

(100-100)

96.6

(95.3-98.4)

95.4

(93.8-97.7)

100

(100-100)

98.0

(97.2-99.1)

3

100

98.3

97.6

100

99.0

4

100

95.0

93.4

100

97.0

5

100

98.3

97.6

100

99.0

CI: Confidence Interval.

Table 4

Performance of HIV kits with Seroconversion Panel Sera (Lot# RP-018)

Panel member

Bleed day

J Mitra & Co. Pvt. Ltd

Span Diag. Ltd.

Transasia Bio-Medicals Ltd.

Bio-Rad

Biomerieux

  

MICROLISA HIV

ENZAIDS HIV 1 + 2

ERBALISA

GENSCREEN

VIRONOSTICA

  

(S/Co)

(S/Co)

(S/Co)

(S/Co)

S/Co)

1

0

0.022

0.235

0.258

0.224

0.238

2

3

0.007

0.162

0.183

0.221

0.15

3

9

0.007

0.177

0.19

0.238

0.112

4

13

0.015

0.148

0.171

0.238

0.254

5

16

0.015

0.162

0.167

2.425

0.233

6

20

0.007

0.162

0.171

2.888

0.258

7

29

0.65

0.877

0.65

3.291

2.002

8

51

6.723

4.401

3.806

9.396

2.265

9

56

7.511

2.523

2.665

12.724

2.436

10

58

7.285

3.65

4.182

14.224

2.967

11

63

5.226

5.549

4.601

16.388

2.95

12

65

7.635

5.069

5.247

17.993

3.245

13

70

6.211

5.339

5.277

19.828

3.026

14

72

6.073

6.419

5.787

20.91

4.513

15

77

6.825

3.552

3.76

21.045

4.189

16

79

7.182

4.639

4.95

22.425

4.472

17

84

7.328

4.177

4.563

22.993

5.386

18

86

7.81

3.953

4.19

22.037

5.729

19

91

5.401

3.639

4.224

23.187

4.72

20

93

7.606

3.964

4.285

23.575

5.604

21

98

7.372

5.141

5.635

23.56

5.976

22

100

6.467

3.469

3.798

24.007

5.988

23

112

7.562

4.372

4.688

29.881

6.254

24

114

7.993

3.776

4.213

24.91

6.537

25

133

8.825

7.415

7.988

24.858

6.625

S/Co = Sample/cut of.

Discussion

ELISA is the type of test most commonly used for detection of HIV particularly for large numbers of specimens [2]. But the discordance between the results of different ELISA kits as well as in different lots of the same kit (particularly false positive rates), as evident in this study, highlights an important problem of potentially causing stress to falsely-positive individuals and may also lead to additional expenses [13, 14]. Hence, evaluation of diagnostic ELISA kits gains importance for ensuring the availability of suitable kits with better performance in terms of recommended sensitivity and specificity [11], as in case of blood bank testing where a high degree of sensitivity is also recommended for choosing the testing kit [11]. Better performance, comparatively, was observed for ERBALISA kits with 100% efficiency in 4 out of 5 batches (mean efficiency 99.8%). Microlisa and Enzaids each showed mean efficiency of 99.6%. The performance of Genscreen and Vironostika was compromised in terms of specificity, PPV and efficiency as these kits give few false positive results. The PPV as estimated based on the composition of panel sera will change according to the prevalence in the targeted population to be tested [9]. Thus, in an Indian scenario with 0.27% HIV prevalence [15], the estimated PPV of the HIV ELISA kits would be 40.30% for both J.Mitra & Co. Pvt. Ltd. and Span Diagnostics Ltd., 57.40% for Transasia Bio-Medicals Ltd. and 14.40% for Bio-Rad Laboratories. The performance of all five kits in terms of NPV favors their use as a primary screening assay for HIV infection. Unique combination of simultaneous Ag-Ab assay gives better performance in a seroconversion panel as well as reducing the testing window period by 3.82 days on average [6]. In this study, Genscreen and Vironostika, both 4th generation assays, outperformed the 3rd generation ELISA by reducing the window by 35 and 22 days respectively in the seroconversion panel. Genscreen showed better performance and identified early seroconversion than Vironostika in another study [16]. The 3rd generation kits demonstrated efficiency ranging from 98.6 to 99.8%. Though the efficiency of the 4th generation assays is lower, the high sensitivity demonstrated by the kits may favor them for HIV screening purposes, because of their early detection by about 3 weeks post infection. Although lot to lot variation is evident in the study it is not statistically significant (p > 0.05), but kits with specificity level <98% are not recommended for diagnosis of HIV infection in India according to the national guideline [11].

The panel size is small with 100 members and one seroconversion panel only. Kit evaluation with small panel size may be valuable where studies are limited [17]. Use of a range of seroconversion panels is essential to test the biological differences in the timing of appearance of different antibodies to specific antigens in the host response to the various HIV antigens in a range of individuals. A more robust assessment requires the testing of, for example, 10 different seroconversion panels [18, 19].

Conclusion

With 100% sensitivity, both the 3rd and 4th generation commercial ELISA kits seem are appropriate as screening assay for detection of HIV infection. Earlier detection post HIV infection favors the use of 4th generation kits for ensuring HIV free blood for transfusion. The lot to lot variation in terms of specificity warrants batch pre-acceptance testing of all new lots or batches of commercially available ELISA kits in India to ensure that new batches perform as well as previous ones.

Abbreviations

AIDS: 

Acquired Immuno Deficiency Syndrome

CDSCO: 

Central Drug Standard Control Organization

ELISA: 

Enzyme Linked Immunosorbent Assay

HIV: 

Human Immunodeficiency Virus

NPV: 

Negative Predictive Value

PPV: 

Positive Predictive Value

USFDA: 

United States Food and Drug Administration.

Declarations

Acknowledgements

We acknowledge NACO, New Delhi and West Bengal State AIDS Prevention and Control Society for partially supporting the study.

Authors’ Affiliations

(1)
National HIV Reference Laboratory, National Institute of Cholera and Enteric Diseases

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Copyright

© Nandi et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

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