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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2007  |  Volume : 18  |  Issue : 4  |  Page : 523-531
Evaluation of the available anti-HCV antibody Detection Tests and RT-PCR assay in the Diagnosis of Hepatitis C Virus Infection


1 Consultant Internal Medicine and Nephrology, Al-Noor Specialist Hospital, Makkah, Saudi Arabia
2 Director of Laboratory Department, Al-Noor Specialist Hospital, Makkah, Saudi Arabia
3 Professor of Clinical Pathology, Al-Noor Specialist Hospital, Makkah, Saudi Arabia
4 General Surgery Consultant, Al-Noor Specialist Hospital, Makkah, Saudi Arabia
5 Consultant Diabetes, Al-Noor Specialist Hospital, Makkah, Saudi Arabia
6 Prof. Benha Faculty of Medicine, Al-Noor Specialist Hospital, Makkah, Saudi Arabia

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   Abstract 

The aim of the present work is to evaluate the commercially available antibody tests in the diagnosis of hepatitis C virus (HCV) infection by comparing their results with the RT-PCR test. The study included 316 serum samples from three groups: blood donors, patients on maintenance hemodialysis (HD) and patients infected with the human immunodeficiency virus (HIV). Samples were subjected to HCV-antibody detection by ELISA and RIBA tests and HCV­RNA detection by RT-PCR assay. The percentage of infectivity for blood donors was 18.9% by ELISA, 20.8% by RIBA and 23.6% by RT-PCR test. For patients on HD and those positive for HIV, the test positivity was respectively 59.3% and 5.3% by ELISA, 64% and 10.5% by RIBA and, 66.3% and 21% by PCR test. The percentage of false negativity of HCV-Ab by ELISA and RIBA when compared with RT-PCR test was 3.5 and 8.1% for samples blood donors, 17.1 and 25.7% for HD patients and 5.6 and 16.7% for HIV-infected samples, respectively. The false positivity of HCV-Ab by ELISA and RIBA, when compared with RT-PCR, was 5%, 3.9% and zero for blood donors, HD patients and HIV-HCV co-infected cases, respectively. While comparing ELISA with RT-PCR, the false positivity was 10%, 5.9% and zero respectively for blood donors, HD patients and HIV-HCV co-infected cases. Thus, it is very important to screen blood donors, HD patients and HIV-infected patients by using the RT-PCR for HCV-RNA to avoid false negative results.

How to cite this article:
Tashkandy MA, Khodari YA, Ibrahim AM, Dhafar KO, Gazzaz ZJ, Azab BA. Evaluation of the available anti-HCV antibody Detection Tests and RT-PCR assay in the Diagnosis of Hepatitis C Virus Infection. Saudi J Kidney Dis Transpl 2007;18:523-31

How to cite this URL:
Tashkandy MA, Khodari YA, Ibrahim AM, Dhafar KO, Gazzaz ZJ, Azab BA. Evaluation of the available anti-HCV antibody Detection Tests and RT-PCR assay in the Diagnosis of Hepatitis C Virus Infection. Saudi J Kidney Dis Transpl [serial online] 2007 [cited 2018 Jul 21];18:523-31. Available from: http://www.sjkdt.org/text.asp?2007/18/4/523/36506

   Introduction Top


Hepatitis C virus (HCV) is recognized as a major threat to global public health. [1],[2] Although advances have been made, a reliable culture system for HCV is not yet available. [3] Labora­tory assays that are available for the diagnosis and management of HCV infection include: (a) serological tests to detect HCV antibodies (enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA), (b) molecular tests to detect and quantitate HCV-RNA and (c) genotyping. [4] Only 50 to 70% of patients with acute infection who develop symptoms, will have detectable anti­bodies at that time, although 90% will have measurable antibodies after a period of three months. [5],[6] Serological assays detect HCV antibodies that indicate present or previous infection, but they cannot discriminate acute from chronic or resolved infection. [7] An addi­tional drawback is that false positive results may occur in patients with autoimmune diseases and in neonates born to mothers with chronic HCV infection. [8] On the other hand, false negative results are most common very early after infection (it takes 6-8 weeks for third generation ELISA to yield positive results versus 2-3 weeks for PCR) or in patients who have an impaired immune system e.g. patients with human immuno-deficiency virus (HIV) infection, those on hemodialysis (HD) and patients on chemo­therapy drugs. [9],[10],[11]

The aim of this work was to evaluate results of HIV-antibody testing in the diagnosis of HCV infection among blood donors, HD and HIV-infected patients and compare the results with RT-PCR test for the diagnosis of HCV infection.


   Materials and Methods Top


The current study was carried out on 316 serum samples divided into three groups: Group I: 106 samples from male blood donors, as controls, Group II: 172 samples from patients on HD selected from the renal and hypertension unit (RHU) and Group III: 38 samples from HIV-positive (by screening HIV 1, 2 Ag/Ab ELISA test and confirmed by Western Blot test) detected from blood donors and the out-patient clinic.

All samples were collected at the Immuno­logy and Serology Department at the Al-Noor Specialist Hospital, Makkah. We excluded all samples from patients with diabetes or other endocrine diseases and autoimmune diseases. Also hemolysed, lipemic and jaundiced sam­ples were excluded. All samples were aliqua­ted into two portions; one was kept at 70° C until processing for RT-PCR and the other was subjected to HCV antibody detection by ELISA and RIBA methods.


   Methods Top


Screening enzyme linked immunosorbent assay (ELISA)

The ABBOTT Murex anti-HCV ELISA third generation test was used for detection of antibodies to HCV in serum samples. [12]

Recombinant Immunoblot assay (RIBA)

The RIBA test was performed using a line immunoassay (LIA) for the detection of antibodies to HCV in serum samples using commercial kit of INNo-LIA HCVAb III update -INNOGENTICS-Belgium, which is a third generation line immunoassay [13] [Figure - 1],[Figure - 2].

RT-PCR assay

Viral RNA was extracted from 200 µl of each sample, using the High Pure Viral Nucleic Acid reagent set (Roche Molecular Biochemicals). Nucleic acids were extracted in 50 µl of nuclease-free water. At least one positive and one negative control were processed in parallel with each batch of samples. We used primers, which identify 284 bp, sequence of the highly conserved 5´ untranslated region of the HCV genome [Figure - 3]. [14],[15],[16]


   Results Top


The positivity rates for HCV infection with the various tests used among blood donors were: 18.9 % (20 out of 106) by ELISA, 20.8 % (22 out of 106) by RIBA and 23.6 % (25 out of 106) by RT-PCR. For patients on HD, they were 59.3 % (102 out of 172) by ELISA, 64 % (110 out of 172) by RIBA and 66.3 % (114 out of 172) by RT-PCR. Among the HIV-Positive patients, the positivity rates were 5.3% (2 out of 38) by ELISA, 10.5% (4 out of 38) by RIBA and 21.1% (8 out of 38) by the RT-PCR technique [Table - 1] and [Figure - 4].

The percentage of false negativity of HCV­Ab ELISA screening test compared with RIBA and RT-PCR was 3.5 % and 8.1% respectively among blood donors. Among patients on HD, the false negativity was 7.1% when repeated by RIBA and 25.7% when compared with RT-PCR. The HIV-HCV infected serum samples showed 5.6 and 16.7% false negative ELISA results when compared with RIBA and RT-PCR respectively [Table - 2] and [Figure - 5].

The percentage of false positivity of HCV­Ab ELISA test when compared with RIBA and RT-PCR, was respectively 5% and 10% for blood donors, 3.9% and 5.9% for HD patients and zero for HIV-HCV infected patients [Table - 3] and [Figure - 6].


   Discussion Top


Clinical and therapeutic decisions for HCV infection depend on factors that include documentation of past infection as well as identification of presence or absence of certain genotypes of the virus. [17] To aid in decision-making, two diagnostic blood tests are available for HCV infection; the anti­body tests (ELISA and RIBA) and the poly­merase chain reaction for RNA detection. [18] The presence of HCV antibodies suggests prior exposure to HCV but does not confer immunity, while PCR-RNA detection indi­cates that the patient has detectable levels of HCV in his blood. [19]

As the levels of HCV-RNA fluctuate above and below the threshold for detection by PCR, [20] our data comparing the results of these tests in diagnosis of HCV infection, showed marked discrepancy in infection percentage within the three groups studied.

In a study performed in India by Baheti et al in 2000, [21] the percentage of seroprevalence of anti-HCV antibodies was studied among healthy blood donor volunteers and high­risk individuals. They found that, out of 99 health-care workers from various departments, a total of 9.09% were positive for anti HCV­Ab; they included dental surgeons (20%), nursing staff (12.9%), surgeons (11.1%) and physicians (4.7%), who were infected in descending order of frequency. They did not find any infection in pathologists and lab workers. The prevalence of anti-HCV Ab in patients on HD was 38.09% and among healthy blood donors was 2.4%. These values suggest that there is a higher prevalence of HCV infectivity in Saudi Arabia among these groups and this may be due to multinational nature and pooling of population, especially in Makkah.

The percentage of false negativity of HCV­Ab ELISA screening compared to RIBA and RT-PCR test among blood donors was 3.5% (3 out of 86) and 8.1% (7 out of 86) respectively. This false negativity may be due to the presence of low levels of anti­bodies in blood (despite viremia) during the HCV-window period. This has been docu­mented by McCoy et al in 1999, [22] who reported that 90% of infected people will produce antibodies within three months of exposure and 10% take an even longer time to produce the antibodies and thus, the test needs to be repeated six months after exposure.

Also, the increased false negativity for anti­body tests (ELISA and RIBA) compared with PCR-RNA detection in HD and HIV samples may be due to weaker and delayed response of these immunocompromized pa­tients to produce antibodies. Fernandez et al, in 1996 [23] using third generation anti-HCV EIA test found that 12.9% of HD patients had detectable serum HCV-RNA despite their anti-HCV negativity. Hanuka et al, in 2002 [24] found that 9% of seronegative dialysis patients were HCV-RNA positive with low viral load. These data question the isolation criteria that are used presently in some HD units to prevent HCV transmission. Although periodic anti-HCV determinations remain mandatory, their negativity does not discard the possibility of infection.

The current study shows an interesting and important data on comparing results for false negativity among patients with HIV (16.7%), which was lower than in HD patients (25.7%). This may be due to the higher prevalence of HCV infection among dialysis patients (66.3%) than HIV patients (21%), as well as higher chance of viral transmission through dialysis machines. [4] These results are docu­mented by Steven Kolker in 2000, [25] who reported that third generation ELISA and RIBA assays pick up only half of HCV­RNA positive patients co-infected with HIV.

The false positivity of ELISA screening test for HCV-Ab as compared with RIBA was respectively 5%, 3.9% and zero for blood donors, HD patients and HIV-HCV co­infected cases (the two cases with HIV who tested positive for HCV-Ab were also positive RIBA). When ELISA was compared with RT­PCR, the false positivity was 10%, 5.9% and zero among blood donors, HD and HIV cases respectively. This suggests a high specificity of RT-PCR to exclude HCV-viremia inspite of presence of non-specific antibodies. Martins et al in 1994, [26] using second generation ELISA, reported that HCV-viremia was present in 76.6% of the anti-HCV positive blood donors, (23.4% false positive) in central Brazil.

The presence of HCV-Ab in the absence of viremia in blood donors, may be due to a previous infection and that the person is immune at the time of examination. [27] Among immune compromised patients, the percentage of false antibody positivity is less since synthesis of antibodies in these cases indi­cates the presence high viremia to stimulate their depressed immune system. However, the presence of false positivity by RT-RNA may be due to the presence of non-specific antibodies detected by ELISA screening test. [28],[29]

The high percentage of false negativity for HCV-antibody detection by ELISA may be due to the immunosuppression that charac­terizes HD and HIV patients or it may be due to the absence of detectable anti-HCV anti­body levels during the window stage of infection. The high degree of false negativity associated with ELISA screening for HCV­Ab among blood donors is a big problem and leads to increased risk of transfusing contaminated blood. On the other hand, false positivity leads to increased wastage of otherwise the safe units of blood, especially those belonging to rare blood groups, apart from causing anxiety to the donor.

Thus, it is very important to screen blood donors, HD and HIV patients by using the RT-PCR for HCV RNA to avoid the occur­rence of false negative results of HCV anti­body detection tests. To avoid false positive results, we should repeat RT-PCR twice every year and if it remains negative, this means that the patient is free from viremia. However, for HD patients, we recommend the use of highly specific and sensitive tests for detection of any virus in the blood to control and prevent infection with the HCV.

 
   References Top

1.Lauer GM, Walker BD. Hepatitis C virus infection. N Eng J Med 2001;345: 41-52.  Back to cited text no. 1    
2.Booth JC, O'Grady J, Neuberger J. Clinical guidelines of the management of hepatitis C. Gut 2001;49:1-21.  Back to cited text no. 2    
3.Bendinelli M, Pistello M, Maggi F, Vatteroni M. Blood-born hepatitis viruses: Hepatitis B, C, D and G viruses and TT virus, Clinical virology manual, 3rd ed. ASM Press: Washington, DC; 2000.  Back to cited text no. 3    
4.Sandra SR. Laboratory Assay for diagnosis and management of hepatitis C virus infection. J Clin Microbiol 2002; 40:4407-12.  Back to cited text no. 4    
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6.Marison G, Pirola R, Lacarini S, et al. Acute Hepatitis C. Australian Family Physician (Special Issue) 1999;28:46­50.  Back to cited text no. 6    
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10.Fabrizi F, Poordad FF, Martin P. Hepatitis C infection and the patient with end­stage renal disease. Hepatology 2002; 36:3-10.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Stuyver L, Claeys H, Wyseur A, et al. Hepatitis C virus in a hemodialysis unit: Molecular evidence for nasocomial transmission. Kidney Int 1996;49:889-­95.  Back to cited text no. 11  [PUBMED]  
12.Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A non-B hepatitis. Science 1989;244:362-4.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]
13.Bukh J, Wantzin P, Krogsgaard K, Knudsen F, Purcell RH, Miller RH. High prevalence of hepatitis C virus (HCV) RNA in dialysis patients: Failure of commercially available antibody tests to identify a significant number of patients with HCV infection. Copenhagen Dialysis HCV Study Group. J Infect Dis 1993;168:1343-8.  Back to cited text no. 13    
14.Kleter GE, Doorn LJ, Brouwer JT, Schalm SW, Heijlink RA, Quint WG. Sequence analysis of the 5'untranslated region in isolates of at least four genotypes of hepatitis C virus in the Netherlands. J Clin Microbiol 1994;32:306-10.  Back to cited text no. 14    
15.Simmonds, P, McOmish F, Yap PL, et al. Sequence variability in the 5' non - coding region of hepatitis C virus: Identification of a new virus type and restrictions on sequence diversity. J Gen Virol 1993;74: 661-8.  Back to cited text no. 15    
16.Bukh J, Purcell RH, Miller RH. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc Natl Acad Sci USA 1992;89:187-91.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Alberti A, Noventa F, Benvegnu L, Boccato S, Gatta A. Prevalence of liver disease in a population of a symptomatic persons with hepatitis C virus infection. Ann Intern Med 2002;137:961-4.  Back to cited text no. 17    
18.Prince AM, Scheffel JW, Moore B. A search for hepatitis C virus polymerase chain reaction-positive but seronegative subjects among blood donors with elevated alanine aminotransferase. Transfusion 1997;37:211-4  Back to cited text no. 18  [PUBMED]  
19.Australian-Hepatitis Council. Post test information for hepatitis C. 1999; Contact, 99 July.  Back to cited text no. 19    
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21.Baheti R, Gehlot RS, Baheti R. Seroprevalence of anti HCV Ab in healthy voluntary blood donors and in high risk individuals. J Indian Acad Clin Med. 2000;1:230.  Back to cited text no. 21    
22.McCoy R, Watson K, Kosky M. A guide to diagnosis. Aust Fam Physician 1999;28S:19-23.  Back to cited text no. 22    
23.Fernandez JL, Noenri DL, Rodolo V, Berridi J. Serum hepatitis C virus in anti­HCV negative haemodialysis patients. Dial Transplant 1996;25:14.  Back to cited text no. 23    
24.Hanuka N, Sikuler E, Tovbin D, et al. Hepatitis C virus infection in renal failure patients in the absence of anti hepatitis C virus antibodies. J Viral Hepatol 2002; 9:144-5.  Back to cited text no. 24    
25.Kolker S. Screening for hepatitis C. in patients coinfected with HIV. Screening for hepatitis C- Department Laboratory Medicine University of California: San Francisco; July 2000.  Back to cited text no. 25    
26.Martins RM, Vandenborght BO, Rouzere CD, et al. Anti-HCV related to HCV PCR and risk factors analysis in a donor population of Central Brazil. Rev Inst Med Trop Sao Paulo 1994;36:501.  Back to cited text no. 26    
27.Murphy El, Bryzmans Williams AE, Co-Chien H, et al. Demographic deter­minants of hepatitis C virus sero­prevalence among blood donors. JAMA 1996;257:995-1000.  Back to cited text no. 27    
28.Furusyo N, Hayasi J, Kanamoto- Tanaka Y, et al. Liver damage in hemodialysis patients with hepatitis C virus viremia: A prospective 10 years study. Dig Dis Sci 2000;45:2221-8.  Back to cited text no. 28    
29.Sancez Quijano A, Andreu J, Gavilan F, etal. Influence of uman immunodeficiency virus type 1 infection of the natural course of chronic parenteral acquired hepatitis C. Eur J clin Microbiol Infect Dis 1995;14:949-53.  Back to cited text no. 29    

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Correspondence Address:
Zohair J Gazzaz
Consultant Diabetes, Health Research Center Director, Al-Noor Specialist Hospital, Makkah
Saudi Arabia
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    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3]

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