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Year : 2019  |  Volume : 17  |  Issue : 4  |  Page : 108-111

A comparative study for screening human immunodeficiency virus 1/human immunodeficiency virus 2 with third-generation and fourth-generation human immunodeficiency virus ELISA kits in donors from a tertiary care hospital in Northeast India

1 Department of Pathology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
2 Department of Microbiology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
3 Department of Cancer Epidemiology and Biostatistics, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
4 Department of Surgical Oncology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
5 Department of Gynecologic Oncology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India

Date of Submission18-Sep-2018
Date of Decision22-Aug-2019
Date of Acceptance23-Aug-2019
Date of Web Publication12-Dec-2019

Correspondence Address:
Dr. Rashmisnata Barman
Department of Microbiology, Dr. B. Borooah Cancer Institute, Guwahati, Assam
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cmi.cmi_36_18

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Background: Blood transfusion is the most effective means of transmission of human immunodeficiency virus (HIV). The use of fourth-generation ELISA kits has significantly reduced the detection period of the viruses to its window period within 2–4 weeks. Objective: To analyze and compare the profile of blood donors who sero-converted to HIV positive. Materials and Methods: Blood donors reporting to the blood bank attached to a regional cancer center from January to December 2017 were included. The study was conducted in two phases. Phase 1 consists of random testing for HIV1/HIV2 reactive and nonreactive samples from clinical blood donors in the age group of 18–50 years. Phase 2 consists of confirmatory test with Western blot technique. Results: The fourth-generation kit confirmed 139 nonreactive out of 140 samples tested after running the samples with independent confirmatory test, whereas the third-generation ELISA test detected two HIV-reactive samples which were confirmed to be nonreactive by Western blot. Conclusions: For better clinical diagnosis and early detection of HIV, the use of fourth-generation test kits for screening of HIV infection is recommended.

Keywords: AIDS, blood donors, human immunodeficiency virus seropositivity, Western blot

How to cite this article:
Sarma A, Barman R, Kalita C, Sharma JD, Krishnatreya M, Talukdar A, Kalita M, Kataki AC. A comparative study for screening human immunodeficiency virus 1/human immunodeficiency virus 2 with third-generation and fourth-generation human immunodeficiency virus ELISA kits in donors from a tertiary care hospital in Northeast India. Curr Med Issues 2019;17:108-11

How to cite this URL:
Sarma A, Barman R, Kalita C, Sharma JD, Krishnatreya M, Talukdar A, Kalita M, Kataki AC. A comparative study for screening human immunodeficiency virus 1/human immunodeficiency virus 2 with third-generation and fourth-generation human immunodeficiency virus ELISA kits in donors from a tertiary care hospital in Northeast India. Curr Med Issues [serial online] 2019 [cited 2020 Oct 29];17:108-11. Available from: https://www.cmijournal.org/text.asp?2019/17/4/108/272797

  Introduction Top

Access to adequate, safe, and quality blood products is vital for any healthcare system, which is the responsibility of the government/national health authority of a country.[1] Transfusion-transmitted infections are major problems associated with blood transfusion.[2] According to the National AIDS Control Organization (NACO), in 2013, the seroprevalence of human immunodeficiency virus (HIV) among blood donors in India was 0.28%. With the frequent improvement in screening procedure and detection techniques, HIV infection rates via direct blood transfusion have been significantly reduced from 8% in the mid-1990s to 1% in 2009.[3]

Serological event states that HIV antigens appear before the development of anti–HIV antibodies are some HIV antibodies in an HIV-infected patient. However, these antigens are lost due to semi-conversion process, and within 1–2 months of the infection, antibodies are developed, which thereby increases the level of antibodies in the serum.[4] The fourth-generation ELISA kit is developed to detect anti-HIV ENV (envelop) antibodies for HIV-1and HIV-2 along with p24 antigens. The detection of p24 antigen significantly reduces the HIV screening capability within the window period from 2 weeks from the day of infection and thus reduces the immunological window period to approximately 16 days. The third-generation test detects antibodies (IgM and IgG) within about 22 days. However, third-generation kits are incapable of detecting the presence of p24 antigens, which increases the time period for the detection of infected blood to 4 weeks from the day of exposure to HIV. The odds of detecting HIV infection during the window period can be improved by the fourth-generation ELISA kit, and the present study compared sensitivity, specificity, and positive predictive values (PPVs) of both third- and fourth-generation ELISA test kits.

  Materials and Methods Top

This study was conducted after approval by the Institutional Research and Ethics Committee. A total of 140 serum samples of voluntary blood donors from the blood bank of a regional cancer center were collected. This was a comparative study of both the commercially available third-generation ELISA kit for the detection of antibody and the fourth-generation ELISA kit for the detection of both p24 antigen and antibody to HIV. Informed consent was obtained from the blood donors for the sample collection and enrolment in the study.

Exclusion criteria – Professional blood donors were also excluded from the study.

Blood sample of 2 ml was collected using a sterile 24G disposable needle and syringe in plain tubes. The sera were separated after centrifugation. All 140 blood donors enrolled in the study were counseled before testing. As per the Drugs and Cosmetics Act (Third Amendment 2001) Government of India, blood donors enrolled in the present study were tested for HIV antibodies using third-generation ELISA kit manufactured by Span Diagnostic Ltd., Kolkata, India, and fourth-generation ELISA kits manufactured by J. Mitra and Co. Pvt. Ltd., New Delhi, India. All the collected samples were tested for HIV1/HIV2 infection. The reactive samples and nonreactive samples were further tested for confirmation via Western blot strips manufactured by J. Mitra and Co. Pvt. Ltd, New Delhi, India. All the usage guidelines, precautionary measures, and observation based on the manufacturer's kit manual were strictly observed, and optical density (OD) obtained from the ELISA reader was manufactured by Robonik India Pvt. Ltd., Thane, India. The OD values which were greater than value of the cutoff value (COV) of the respective kits were considered reactive, and OD values lower than COV were marked nonreactive to screening. The COV was as per the manufacturer's instructions. All the samples with OD in the range of grey zone which OD greater or lower than COV, but in the range of 10% of the COV, were retested according to the manufacturer's guidelines. Grey zone issue was resolved by repeat testing, and if the results were unequivocal, then Western blot test was done. All 140 blood samples were tested for hepatitis B virus (HBV), hepatitis C virus (HCV), malaria, and syphilis separately as per the NACO guideline. The institute where this study has been conducted follows the NACO guidelines for doubtful results. Doubtful results are re-tested using three different kits based on different principles of kit manufacturers. Accordingly, reactive patients are referred to Integrated Counselling and Testing Center for further management.

  Results Top

Of 140 blood donors, the highest number of blood donors included in our study was in the 26–30 years of age group (38/27.14%), and details are shown in [Table 1]. One hundred and twenty-four (88.5%) blood donors included in the present study were males, and 15 (11.43%) were females.
Table 1: The age group distribution of blood donors

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Three samples were reactive for HBV, one was reactive for HCV, and one sample was reactive for syphilis. The samples that were positive for HBV, HCV, and syphilis were nonreactive to the fourth-generation HIV1/HIV2 ELISA kit test, which concluded the absence of no direct cross-reactivity. Furthermore, two false-reactive results obtained by the third-generation HIV1/HIV2 ELISA kit did not overlap the samples tested with reactive HCV, HBV, and syphilis.

Specificity and sensitivity

The specificity and sensitivity analysis was done using free online tool (https://www.medcalc.org/calc/diagnostic_test.php). Of 140 donor's serum samples tested by the third-generation kit manufactured by Span Diagnostic Ltd., Kolkata, India, three HIV-reactive samples were detected of which two turned out to be false positives, which was confirmed by the Western blot test. The fourth-generation kit manufactured by J. Mitra and Co. Pvt. Ltd., New Delhi, India, confirmed 139 nonreactive samples after running the samples with independent confirmatory test and one true-positive test confirmed by Western blot. Specificity of fourth- and third-generation HIV1/HIV2 virus screening kit test was 100% and 98.5%, respectively. The sensitivity of both third- and fourth-generation HIV1/HIV2 ELISA test kits was 100%. The PPV for third-generation HIV1/HIV2 ELISA test kit was 33.3% compared to 100% for fourth-generation test kit.

  Discussion Top

The most common method of transmission of HIV is through blood transfusions, and this is due to the large volume of inoculum injected.[5] In recent years, highly sensitive screening methods have reduced the immunological window for detecting HIV in blood donors. Furthermore, with the establishment of counseling centers that test HIV for free of charge and with full confidentiality, it has improved HIV screening among blood donors. This has increased the number of people who enroll as blood donors and are also interested in the screening of HIV results.[6] This has been made possible with the support of NACO's capacity building plan for blood bank staff and effective counseling of blood donors, by strengthening quality monitoring system and emphasizing the appropriate clinical use of blood. Introduction of External Quality Assurance Scheme EQAS programs with encouragement to move toward accreditation will help in further ensuring transfusion safety.[7] However, some risk still remains as the currently available laboratory tests have reduced the immunological window period, but not eliminated it totally. Moreover, early diagnosis of HIV and treatment of seropositive patients are very important, as immediate initiation of antiretroviral drugs can dramatically reduce HIV transmission and mortality in high prevalence settings.[8]

In the present study, the highest number of blood donors included was in the 26–30 years of age group 38 (27.14%), followed by the 21–25 years of age group 30 (21.42%), and this finding is similar to a study conducted by Sujatha and Vaisakhi.[9] The number of donors in 46–50 years of age group was 3 (2. 14%). The reason for less number of voluntary donors in this age group may be due health-related issues such as hypertension and diabetes mellitus which are usually associated with the people of middle-aged groups. Of 140 voluntary blood donors participating in the present study, 89% were males and only 11% were females. Our findings are similar to those of Gisselquist et al., 2004, who reported that males were 95.4% blood donors and females were 4% of all donors.[10] In the Indian scenario, females are usually malnourished, anemic due to repeated childbirth, so they do not fulfill the screening criterions of blood donors in most of the time.

The comparative study of both the commercially available third- and fourth-generation ELISA kits resulted in different result based on its sensitivity and specificity. In the present study, the ability of the fourth-generation ELISA kit to detect and screen p24 antigen in the serum/plasma was to screen and confirm an additional positive sample in the donated blood units, which was confirmed nonreactive by the third-generation ELISA kit. The appearance of one confirmed HIV-reactive samples per 140 tested donor blood units. However, in our study, the preliminary test with third-generation kit detected two false-reactive samples. Similar studies done by Muthukumar et al. also showed that that the fourth-generation HIV assays yield fewer false-positive and false-negative results than the third-generation HIV assays we tested.[11] The specificity of fourthgeneration kit in the present study were 100% compared to 98.5% specificity of thirdgeneration test kit.

In the present study, samples were also tested based on the observation of possible cross-reactivity for HBV, HCV, and syphilis. Three samples reactive for HBV, one for HCV, and one for syphilis were also tested for possible cross-reactivity of the samples collected. The results obtained showed clear nonreactivity for HIV, which concluded the absence of no direct cross-reactivity observed in the study. However, a study showed that co-infection with HIV and HBV was observed among 0.77% donors, followed by hepatitis B and C coinfection (0.21%) and HIV and HCV coinfection (0.06%).[12]

There are some limitations to the present study. First, the number of samples tested was less. Second, the Western blot test was used for confirmation, but the gold standard for confirmatory test is the nucleic acid amplification test. A good history such as exposure history and risk factor history and fourth-generation HIV ELISA is sufficient for preventing blood-borne viral infections.

  Conclusions Top

Fourth-generation ELISA kit for HIV1/HIV2 is specific and the PPV of the third-generation kit was low. Thus, fourth-generation kit for HIV1/HIV2 is recommended.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Ramani KV, Mavalankar DV, Govil D. Study of blood-transfusion services in Maharashtra and Gujarat states, India. J Health Popul Nutr 2009;27:259-70.  Back to cited text no. 1
National AIDS Control Organization. Assessment of Blood Banks: A Preliminary Report. National AIDS Control Organization; 2016.  Back to cited text no. 2
National AIDS Control Organization. Annual Report NACO2010-11. Available from: http://www.nacoonline.org. [Last accessed on 2018 Jun 18].  Back to cited text no. 3
Allain JP, Laurian Y, Paul DA, Senn D. Serological markers in early stages of human immunodeficiency virus infection in haemophiliacs. Lancet 1986;2:1233-6.  Back to cited text no. 4
Savarit D, De Cock KM, Schutz R, Konate S, Lackritz E, Bondurand A. Risk of HIV infection from transfusion with blood negative for HIV antibody in a West African city. BMJ 1992;305:498-502.  Back to cited text no. 5
Kupek E. Transfusion risk for hepatitis B, hepatitis C and HIV in the state of Santa Catarina, Brazil, 1991-2001. Braz J Infect Dis 2004;8:236-40.  Back to cited text no. 6
National AIDS Control Organization. National Strategic Plan for HIV/AIDS and STI 2017 – 2024. National Aids Control Organization; 2017.  Back to cited text no. 7
Granich RM, Gilks CF, Dye C, De Cock KM, Williams BG. Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: A mathematical model. Lancet 2009;373:48-57.  Back to cited text no. 8
Sujatha K, Vaisakhi K. Comparative study of 3rd generation v/s 4th generation ELISA in blood donors for early diagnosis of HIV in rural population. Int J Curr Microbiol Appl Sci 2017;6:1183-93.  Back to cited text no. 9
Gisselquist D, Potterat JJ, Brody S. HIV transmission during paediatric health care in Sub-Saharan Africa – Risks and evidence. S Afr Med J 2004;94:109-16.  Back to cited text no. 10
Muthukumar A, Alatoom A, Burns S, Ashmore J, Kim A, Emerson B, et al. Comparison of 4th-generation HIV antigen/Antibody combination assay with 3rd-generation HIV antibody assays for the occurrence of false-positive and false-negative results. Lab Med 2015;46:84-9.  Back to cited text no. 11
Fouelifack Ymele F, Keugoung B, Fouedjio JH, Kouam N, Mendibi S, Dongtsa Mabou J. High rates of hepatitis B and C and HIV infections among blood donors in Cameroon: A proposed blood screening algorithm for blood donors in resource-limited settings. J Blood Transfus 2012;2012:458372.  Back to cited text no. 12


  [Table 1]


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