Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 2, 2007, pp. 140-142

Indian Journal of Medical Microbiology, Vol. 25, No. 2, April-June, 2007, pp. 140-142

Brief Communications

Evaluation of commercially available third-generation anti-hepatitis C virus enzyme-linked immunosorbent assay in patients on haemodialysis

V Lakshmi, AK Reddy, KV Dakshinamurty

Departments of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad - 500 082

Correspondence Address: Departments of Microbiology (VL, AKR) and Nephrology (KVD), Nizam’s Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh, India (email lgorthi@hotmail.com)

Date of Submission: 29-Sept-2005
Date of Acceptance: 12-Sept-2006

Code Number: mb07039

Abstract

Keywords: Chronic renal failure, enzyme-linked immunosorbent assay, hepatitis C virus, haemodialysis, synthetic peptides

Chronic renal failure (CRF) patients on hemodialysis (HD) are considered to be a high-risk group for contracting hepatitis C virus (HCV) infection. ^[1] The prevalence of antibodies to HCV (anti-HCV) in HD patients ranges worldwide from 1% in UK to 62% in Portugal ^[1] and is highly variable between different countries and between different centers in the same locality. ^[2]

The diagnosis of HCV infection is currently based on the detection of anti-HCV by enzyme immunoassay (EIA) and it is confirmed by a positive result obtained by an immunoblot assay or by the presence of HCV RNA. ^[3],[4] An improvised third-generation HCV enzyme immunoassay (EIA) with high sensitivity is widely used for screening patients. ^[3] Most of the available third-generation enzyme-linked immunosorbent assay (ELISA) tests for anti-HCV detection are based on either synthetic peptide antigens alone or recombinant protein antigens or a combination of synthetic and recombinant protein antigens of HCV. ^[5] In patients with immunosuppressive conditions, serological response is low and this could lead to negative results in serological assays. ^[6] Chronic renal failure patients on HD represent a particular group for whom sensitivity of antibody screening assays used may be critical. ^[2] The recombinant antigen assays are highly sensitive and can detect even a minute quantity of antibody produced. However, the synthetic peptide EIAs have a low sensitivity and may not be able to detect low levels of antibody produced, as in the case of the immunocompromised HD patients. Restricted antibody reactivity to HCV synthetic peptides has been observed in HCV-infected patients on HD. ^[5],[6]

The aim of this study was to evaluate third-generation anti-HCV ELISA test systems containing either synthetic peptide HCV antigens or recombinant HCV antigens or a combination of synthetic and recombinant antigens in screening for HCV infection in CRF patients on hemodialysis.

Materials and Methods

Sixty-nine chronic renal failure patients undergoing hemodialysis from May 2003 to December 2003 were included in the study. Blood samples were collected from the patients after obtaining informed consent. Serum was separated within two hours after blood sampling. All the serum samples were divided into 0.5 mL aliquots and stored at -20°C and at -70°C for polymerase chain reaction (PCR).

All samples were tested for anti-HCV antibody with three different commercially available third-generation ELISA test systems: a) Ortho HCV 3.0 ELISA test system (Ortho Clinical Diagnostics) using recombinant protein antigens (c22-3, c200 and NS5) originating from four regions of the viral genome (Core, NS3, NS4, NS5); b) UBI HCV EIA 4.0 (Beijing United Biomedical), using synthetic peptides of Core, NS3, NS4 and NS5 regions; and c) SP-NANBASE C-96 3.0 (General Biologicals) using a combination of recombinant (NS3 and NS5 antigens) and synthetic peptides (Core and NS4 antigens).

All the 69 samples were also tested for HCV RNA by RT-PCR with AMPLICOR HCV test version 2.0 (Roche Diagnostics). All the ELISA-positive samples were confirmed by a supplemental anti-HCV immunoblot assay such as CHIRON RIBA HCV 3.0 SIA (RIBA) utilizing ′recombinant HCV′-encoded antigens (c33c and NS5) and ′synthetic HCV′-encoded peptides (c100p, 5-1-1p and c22p).

Results

Sixty-two out of 69 patients were negative by all the three different ELISA test systems. Seven patients were positive in both the ELISA systems containing recombinant antigens. The kit containing only synthetic antigens failed to detect HCV antibodies in these positive patients. Four out of seven anti-HCV positive sera were positive for HCV antibodies in RIBA, with a strong antibody response to NS3 region. All these four cases were also positive for HCV RNA by PCR.

Out of remaining seven anti-HCV positive samples, two were positive in the combination kit and one sample was positive in the recombinant EIA. All these three sera were negative by RIBA and had no HCV RNA, indicating a false positive ELISA result. Consolidated results of all the tests are shown in [Table - 1] and [Table - 2].

The performance parameters of the three ELISA systems with PCR as a gold standard are shown in [Table - 3]. The sensitivity of the synthetic peptide kit is zero and that of both recombinant ELISA kit and kits containing both antigens is 100%. Also, the specificity (67%) and efficiency (87%) of the recombinant antigen kit were higher as compared to the combination kit (67 and 76% respectively).

Discussion

Since the introduction of screening assays for HCV antibodies 13 years ago, major efforts have been made to increase both sensitivity and specificity of the assays. Today the third-generation ELISA is widely used for screening patients infected with HCV. Anti-HCV ELISA is positive in more than 99% of immunocompetent patients with detectable HCV RNA, but these tests may be negative despite chronic HCV replication in patients with profound immunodeficiencies. ^[7] Devasa et al. ^[5] in their study showed that an enzyme immunoassay based on synthetic peptides from HCV allowed the detection of antibodies to HCV in 100% of immunocompetent HCV-infected patients but only in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). As per the published literature, there are no such comparative studies from India.

The intrinsic immunodeficiency in hemodialysis patients leading to an impaired antibody response to HCV antigens has been well described. ^[5],[8] Immune system impairment seems to restrict the spectrum of the antibody isotypes reacting to the synthetic peptides in such states. In our study, four HCV-infected hemodialysis patients were missed using only ′synthetic peptide′-based ELISA. As is well known, synthetic peptides (length of approximately 15-20 amino acids) are deficient in carboxy terminal and thereby are less sensitive, especially in immunocompromised situations like the patients on hemodialysis, where the amount of antibody produced is low. ^[9],[10] On the other hand, the recombinant proteins do not suffer from this deficiency of the carboxy terminal endand hence are more sensitive and they even detect low levels of antibody titer.

Feucht et al. found that NS3 region played an important role in the diagnosis of HCV infection. They detected antibodies against NS3 region in 93.7% of positive samples and found a strong antibody reaction against NS3 region, particularly in early HCV infection. ^[11] In this study, the high sensitivity of the recombinant and the combination kit could probably be attributed to the recombinant NS3 antigen used. Similarly, in the supplemental anti-HCV test (RIBA), all four ELISA-positive samples showed a strong reactivity towards recombinant NS3 antigen.

It has been documented that among the immunocompromised populations, the proportion of false positive results with the third-generation ELISA averages approximately 15%. ^[12] Therefore, one should not rely exclusively on a positive anti-HCV screening test to determine whether a person is infected with HCV. A positive screening test result should be verified with an independent supplemental test (RIBA/PCR) with a high specificity. ^[12] Direct measurement of HCV RNA in the serum of the infected individual remains the gold standard in the diagnosis of HCV infection. ^[9]

All the four ′RIBA confirmed positive′ patients were found to have HCV RNA by the RT-PCR for HCV RNA (true positives). However, the three false positives by ELISA and RIBA negative, though could not be explained, were negative for HCV RNA by RT-PCR. Though the specificity of the pure synthetic peptide kit was 100%, the predictive values and the efficiency are not to the desired level and hence such kits cannot be used in hemodialysis patients.

In conclusion, ELISA systems with recombinant HCV proteins are ideal for screening of HCV-infected hemodialysis patients. The presence of recombinant NS3 antigen in the ELISA cocktail may be crucial for the recognition of HCV-infected hemodialysis patients with low antibody titer.

References

Copyright 2007 - Indian Journal of Medical Microbiology

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