Rapid Immunoassay Alone Is Insufficient for the Detection of Hepatitis C Virus Infection Among High-risk Population

Journal of Viral Hepatitis

R. Firdaus, K. Saha, P. C. Sadhukhan

J Viral Hepat. 2013;20(4):290-293.

Abstract and Introduction

Abstract

Rapid testing for HCV has become a routine practice in resource-limited settings for initial screening. The objective of this study was to evaluate the performance of rapid immunoassay diagnostic test kits for specific and accurate diagnosis of HCV infection among different patient groups in clinical settings of Kolkata, India. Two hundred and fifty-four randomly selected serum samples of 612 samples reported as HCV nonreactive by rapid immunodiagnostic tests were evaluated for HCV antibody, ELISA and HCV RNA testing for confirmatory diagnosis. 15.74% were HCV seropositive by ELISA, and 11.02% were RNA positive by nested RT-PCR. Additionally, 15 HCV-seronegative chronic liver disease patients with high ALT and AST values were screened for HCV RNA, of which five were positive whose viral load ranged from 1.2 × 10² to 4.4 × 10⁶ IU/mL, and the samples belonged to IVDUs and HIV-co-infected individuals. The results showed that HCV rapid immunoassay test cannot be solely relied on as an absolute and accurate diagnostic tool for screening infection of HCV particularly in high-risk group patients such as IVDUs, haemodialysis, thalassaemic and HIV-co-infected patients who need HCV screening frequently.

Introduction

Hepatitis C virus (HCV) infection is a global health problem affecting around 170 million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma.^[1] In India, HCV infection as a cause of acute viral hepatitis has been reported to vary between 0–21% and responsible for 14–26% cases of chronic liver disease.^[1] HCV infection is mostly acquired through transfusion of blood or blood products. A high prevalence of HCV is found in many high-risk groups (HRG) exposed to blood or blood products, like intravenous drug users (IVDUs), patients with paediatric haematologic malignancies and those with thalassaemia and haemophilia. The prevalence of HCV in blood donors in India (1–1.5%) is higher than that in developed countries.^[2]

The laboratory diagnosis of HCV infection is based on the detection of circulating antibodies and viral RNA. The most widely used HCV screening tests are ELISA and HCV RNA by nucleic acid test (NAT), as they are the most appropriate for screening large numbers of specimens on a daily basis.^[3] These assays are technically demanding and require sophisticated laboratory infrastructure. Hence, in developing countries like India, the blood transfusion centres mostly rely on simple, rapid tests for HCV with individual kits as an inexpensive alternative for ELISA or NAT assays. Although the use of simple rapid immunoassays has significantly reduced the risk of HCV transmission, the window period for the detection of recent or new infection among the high-risk group remains a concern. The purpose of our study was to determine the performance of simple rapid immunoassay in patients who belonged to high-risk group such as thalassaemic and haemodialysis patients, who undergo blood transfusions regularly. As no previous literature is available on the contradictory results of the HCV rapid immunoassay, our study aimed to help the clinicians and the laboratory personnel on false-negative results of the rapid immunoassay, so that in future more confirmatory screening tests would be employed in blood transfusion centres to detect accurately the status of the HCV-positive patients.

Material and Methods

Clinical Samples

Blood samples were collected from different liver clinics, haemodialysis centres, HIV Apex Clinic and thalassaemic patients from Kolkata between March 2006 and February 2011. Among the 612 samples received during this period, 254 samples that were hepatitis B and HCV rapid immunoassay nonreactive but having increased levels of AST and ALT in patients with chronic liver diseases were included in this study. This was an IEC-approved study, and informed consent was obtained from the individuals during the collection of blood samples.

Detection of HCV Antibody by Rapid Immunoassay and ELISA

Rapid diagnosis of HCV was performed using fourth-generation HCV TRI-DOT (WHO-GMP certified, sensitivity 100% and specificity 98.9%; J. Mitra, New Delhi, India), as well as with Signal HCV Kit (sensitivity 98% and specificity 99.45%; Span Diagnostics Ltd, Surat, India).^[4] The same sets of samples were tested for the presence of antibodies against HCV core, NS3 and NS5 regions using two commercially available ELISA kits Hepanostika anti-HCV Ultra Kit (Biomerieux, Boxtel, The Netherlands) and HCV Microlisa (J. Mitra) to verify the results. The fourth-generation HCV TRI-DOT utilizes a unique combination of modified HCV antigens conserved across all genotypes from the putative core, NS3, NS4 and NS5 regions of the virus to selectively identify all genotypes of HCV in human serum/plasma with a high degree of sensitivity and specificity.

Detection and Quantitative Estimation of HCV RNA

The viral RNA was extracted using QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. HCV viral RNA was detected by nested RT-PCR based on 5' noncoding region (5' NCR) of HCV genome according to Saha et al..^[5] Briefly, the first-round RT-PCR was performed in 20-μL total reaction volume containing 2 μL of isolated RNA with outer forward primer (PS1) 5'-ACTGTCTTCACGCAGAAAGCGTCTAGCCAT-3' and outer reverse primer (PA1) 5'-CGAGACCTCCCGGGGCACTCGCAAGCACCC-3'. The second-round nested PCR was performed with inner forward primer (PS2) 5'-ACGCAGAAAGCGTCTAGCCATGGCGTTAGT-3' and inner reverse primer (PA2) 5'-TCCCGGGGCACTCGCAAGCACCCTATCAGG-3'. The primers were selected/used in this nested RT-PCR from highly conserved domains within the 5' NCR region of the HCV genome according to Bukh et al.,^[6] which is well conserved across all the six HCV genotypes. A positive band at 256 bp in 1.5% agarose gel stained with ethidium bromide was observed in gel documentation system (Bio-Rad Laboratories, Hercules, CA, USA) for HCV RNA-positive samples. Quantitative hepatitis C viral RNA was determined using ABI real time RT-PCR kit (AgPath-IDTM One-Step RT-PCR kit, Applied Biosystems, Foster City, CA, USA). The HCV primer and probe sequences were directed against the 5' noncoding region of the HCV genome.

DNA Sequencing and Genotyping

Nested RT-PCR-amplified 256-bp amplicon of 5' NCR of HCV genome was gel-purified and directly used for DNA sequencing analysis in an automated DNA Sequencer, model 3130XL (Applied Biosystems) using Big Dye terminator 3.1 kit (Applied Biosystems). The genotypes of the sequences obtained were determined using the NCBI genotyping tool.^[7]

Statistical Analysis

Mean and median values were calculated. The P-values ≤ 0.05 were considered statistically significant.

Results and Discussion

The study involved 612 patients who were screened for HCV infection using rapid immunoassay kits. Seventy-nine per cent of the patients belonged to 40–60 age group, of which 74% of the patients were men and 26% women.

All serum samples selected in this study were evaluated using two different rapid HCV immunodetection kits, namely TRI-DOT and Signal HCV. Of the total 612 samples, 254 (41.50%) reported as HCV nonreactive were screened for this study. Furthermore, of the 254 samples, 40 (15.74%) were seroreactive by both the ELISA methods. All the seropositive samples were screened for HCV RNA detection; among them, 28 (70%) were HCV RNA positive.

Fifteen-ELISA-nonreactive samples from HRG with a history of chronic liver disease and high AST and ALT values were processed for HCV RNA detection. Five samples were HCV RNA positive, of which three were IVDUs and other two were HIV-co-infected patients with a history of surgery and blood transfusion (). All these HCV RNA-positive samples were further processed for viral quantitation and genotyping. It was found that the viral load ranged from 1.2 × 10² to 4.4 × 10⁶ IU/mL and four were genotype 3, whereas one was genotype 1 (). The median ALT value was calculated at 45 IU/mL (min. 15.55, max. 111.32 IU/mL) and AST at 109.63 IU/mL, respectively (min. 60.23, max. 293.36 IU/mL) (Table 1).

From our results, we could surmise that HCV rapid immunoassay gave false-negative results in patients belonging to high-risk group especially in IVDUs, haemodialysis, thalassaemic, and HIV-co-infected patients. 11.02% of samples marked as rapid immunoassay nonreactive were in fact HCV RNA positive as determined by NAT (HCV RNA) assays. Results obtained were compared by additionally testing the samples using ELISAs and NAT. Two different ELISA kits were used to compare the specificity of the results. Forty (15.74%) samples were seropositive by both the HCV ELISA kits. In addition, the findings showed that of 15 ELISA-seronegative samples, five were HCV RNA positive, that is, the patients were active carriers of the HCV infection. All the seronegative samples that were HCV RNA positive belonged to patients in high-risk group. One of the striking observations was that even though these patients were asymptomatic and declared as HCV nonreactive by rapid immunoassay, they had high levels of ALT and AST values that indicated a dysfunction in their liver.

Our genotyping data showed that of the HCV RNA-positive patients, 80% were infected with genotype 3. The viral load in patients with genotype 3 was significantly lower than in those infected with genotype 1. Diagnosis of HCV in these cases is difficult, as these patients cannot produce sufficient anti-HCV antibodies because of immunosuppression.^[8] Additionally, false-negative results could be attributed to genetic heterogeneity, which could affect the serological response. The most prevalent HCV strain in India is genotype 3,^[9] which is a slow-growing variant, and therefore, the probability of false negatives for this genotype may increase in rapid immunoassay tests. Although HCV rapid immunoassays are routinely used in practically all laboratories, our results showed for the first time that many of the samples declared as HCV rapid immunoassay nonreactive in initial screening were in fact HCV RNA positive, that is, the patients were active carriers of the infection. This shows that rapid tests might give false-negative results particularly for patients belonging to HRG. Missing positivity in immunoassays occurs mainly in immunocompromised patients who fail to clear away the antigens or in patients with autoimmune disorders, hyperglobulinemia or in low-risk blood donors who donate blood frequently. In these cases, confirmatory HCV tests by nucleic acid amplification test (NAT) assays remain the method of choice. The presence of HCV RNA in a patient's serum confirms the active state of infection.

To conclude, the present study highlights that HCV rapid immunoassay tests should not relied upon as the sole criterion for screening patients in high-risk group. In these cases, confirmatory methods should be deployed for HCV detection, so that the rate of false-negative results can be scaled down, and the patients can be effectively screened and put on medication as soon as possible. Although this study is in a preliminary stage, it aims to provide useful inputs to scientific community and the policy makers to improve the existing infrastructure in screening patients for HCV.

References

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