Laboratory diagnosis of viral hepatitis C Laboratory diagnosis of viral hepatitis C The Sultan Qaboos University Hospital experience *Said H S Al Dhahry1, Jameel C Nograles2, Situsekara M W W B Rajapakse2, Fadhila S S Al Toqi2, Geraldine Z Kaminski2 SQU JOURNAL FOR SCIENTIFIC RESEARCH: MEDICAL SCIENCES 2003 VOL 5, NO. –2, 5–20 ©SULTAN QABOOS UNIVERSIT Y ABSTRAC T. Objectives: A retrospective study was carried out to assess the performance of hepatitis C diagnostic assays in our labora- tory, and to determine the prevalence of hepatitis C among blood donors at the Sultan Qaboos University Hospital. Methods: From 1991 to 2001, approximately 55,000 serum samples collected from blood donors and patients were submitted to our laboratory for testing. All sera were screened for antibodies to hepatitis C virus (HCV) by three successive generations of the enzyme-linked immunosorbent assay (ELISA). Anti-HCV positive sera were further tested by recombinant immunoblot assay (RIBA). Reverse-transcriptase polymerase chain reaction (RT- PCR) for HCV RNA was carried out on a limited number (241) of ELISA positive samples. Results: Out of 30012 samples from blood donors that were screened for anti-HCV, 272 (0.91%) were positive. Of these, 46.5% were confirmed positive by RIBA. The proportion of patient sera that were confirmed positive varied from 95% among intravenous drug users to 81% in patients with hepatitis to 70% in those with haemoglobinopathies. HCV RNA was detected in 67%, 6%, and 0% of the RIBA positive, indeterminate and negative samples respectively. Conclusions: Based on RIBA, the prevalence of anti-HCV among blood donors in Oman is close to 0.5%. In our experience, RIBA-positivity is predictive of HCV infection in two thirds of subjects, and HCV infection is highly unlikely in those who are RIBA-negative. The experience at SQUH with three types of HCV assays has enabled the laboratory to develop a test algorithm, starting with screening anti-HCV ELISA. Key words: hepatitis C virus, ELISA, RIBA, polymerase chain reaction. 1Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, P O Box 35, Al Khod, Muscat-123, Sultanate of Oman 2Department of Microbiology and Immunology, Sultan Qaboos University Hospital, P O Box 38, Al Khod-123, Muscat, Sultanate of Oman *To whom correspondence should be addressed. Email: sdhahry@squ.edu.om HEPATITIS C VIRUS (HCV) IS A BLOOD-BORNE pathogen that appears to be endemic in most parts of the world. It is estimated by the World Health Organization that there are 70 million HCV- infected persons worldwide.1 Currently, HCV is the lead- ing cause of post-transfusion hepatitis and end-stage liver disease requiring liver transplantation.2 The disease it causes is characterised by silent onset, a high rate of viral persistence, and the potential for development of chronic liver disease, ranging from chronic hepatitis to cirrhosis and occasionally hepatocellular carcinoma.3 The discovery of the genome of HCV in 989 by Choo et al4 paved the way for development of serological and molecular assays for viral hepatitis C. In the first genera- tion of an enzyme-linked immunosorbent assay (ELISA), wells of microtitre plates were coated with purified recombinant antigen c00-3 that was derived from the non-structural 4 (NS4) region of the HCV genome [Figure ]. However, ELISA- was associated with a high percentage (50% to 70%) of false positive results among low-risk blood donors and in the presence of hyperglob- ulinemia.5 Thus, second-generation anti-HCV ELISAs were developed. ELISA-2 by Ortho Diagnostics con- tained recombinant antigens from the core (c22-3), NS3 region (c33c), and NS4 region (c00-3) as well as a part of c00-3, named 5-- [Figure ]. Third generation anti- HCV ELISA was introduced in Europe in 993 and in the USA in 996. In addition to the antigens of ELISA-2, third-generation anti-HCV ELISA uses an antigen of the NS5 region of the viral genome. However, synthetic pep- Said dhahri �����������������������������������: ���������������������������� ������������������������������������������������������������������������� ��������:�����:�������������������������������������������������������������������������������������� �������������������������������������������������������������������������������:-�������������������� ����������������������������������������������������������ELISA.������������������������������������ RIBA.������������������������������������������������)RT-PCR(.�������:������������������������������� �����)���� (%�������������������������������%����������������������RIBA.����������������������������� ������������������������������������������%��� %���%��������.������������������������������������������ �������������������RIBA���������%�� %�� %��������.������:������������������������������������������ ��������������������������������������� %������RIBA.�������������������������������������������������������� ��������������������RIBA.������������ :����� ������ ��������� ������ ������������������������������� 6 D H A H R Y E T A L tide antigens (c22 and c-00) replaced recombinant anti- gens of ELISA-2 [Table , Figure ]. Other manufactures, for example Abbott Diagnostics, used recombinant anti- gens derived from the same regions of HCV genome. Despite increased sensitivity and specificity with each generation of ELISA, false-positive antibody results con- tinue to be observed, particularly among low-risk blood donors.6 Thus, supplemental or confirmatory assays were developed in parallel with ELISA. The recom- binant immunoblot assay (RIBA) has been used exten- sively to confirm presence or absence of antibody to HCV epitopes. In RIBA recombinant or peptide HCV antigens are blotted as separate bands onto a nitrocel- lulose strip flanked by a weak-positive (Level I) and a moderately positive (Level II) strip control [Figure 2]. Although technically more demanding than ELISA, the RIBA identifies antibodies to individual HCV antigens and therefore has higher specificity than ELISA.7 Since ELISA and RIBA are antibody tests, positiv- ity of either one or both does not necessarily indicate current HCV infection as patients who have recovered from infection may remain anti-HCV positive for many years.7 Conversely, during seroconversion, antibody tests may be negative.8 The direct molecular qualitative detec- tion of HCV RNA by reverse-transcriptase polymerase chain reaction (RT-PCR) is considered the gold standard for the diagnosis of HCV infection.6 Testing for HCV was introduced at the Sultan Qaboos University Hospital (SQUH) in 99, starting with the first-generation ELISA. In subsequent years, new gen- erations of both ELISA and RIBA were used as they became commercially available. The RT-PCR was intro- duced in the year 2000. We report here our results and experience in testing blood donors, patients suspected to have viral hepatitis, and other subjects from 99 to 200. M A T E R I A L S A N D M E T H O D S Sera. Serum samples for HCV testing were collected from blood donors and patients mainly at SQUH. A few were sent from other hospitals within the capital area Table 1. Antigens incorporated in serological assays (ELISA and RIBA) for hepatitis C. Assay ELISA RIBA First generation c100-3 c100-3 5-1-1 Second generation c100-3 c100-3 c22-3 5-1-1 c33c c22-3 c33c Third generation c100-3 c100-3/5-1-1 (peptide) c200 (c100-3 + c33c) c22-3 (peptide) c22-3 c33c NS5 recombinant antigen NS5 recombinant antigen Figure 1. Genome organization of HCV and antigens licensed for diagnostic use. 7 of Muscat. In 99, approximately ,350 sera were tested. In 992, the figure had trebled, and by 200, it reached 6,500. Over a period of  years, approximately 55,000 serum samples were submitted for HCV testing. Screening for Anti-HCV. All sera were first screened for the presence of antibodies to HCV. In 99, the mic- totitre-based, first-generation ELISA (HCV c00-3 ELISA, Ortho Diagnostic Systems, Raritan, NJ) was used. Between 992 and 996, different formats of second- generation enzyme immunoassays (Abbott Diagnostic Division, Germany) were used in the following chrono- logical order: HCV EIA on Quantum™ instrument, micro particle enzyme immunoassay on IMX,™ and micro parti- cle enzyme immunoassay on Axsym™ analyser. The three analysers were from Abbott laboratories, Diagnostic Division, USA. From 997 to 200, sera were screened by a third-generation micro particle enzyme immunoassay on the Axsym.™ Samples that were initially reactive by ELISA were retested in duplicate, and results were inter- preted according to manufacturers’ instructions. Supplementary Assay. The second-generation recom- binant immunoblot assay (RIBA HCV 2, Chiron Corp., Emeryville, CA) was introduced in our laboratory in mid- 994, and was replaced by third generation assays (RIBA HCV 3, Chiron and HCV Blot, Genelab Diagnostics, Singapore) by the end of 996. All sera that were repeat- edly reactive in ELISA were subjected to supplemental testing by RIBA according to manufacturers’ recom- mendations. In these assays specimens were considered positive if they demonstrated reactivity to two or more antigen bands at an intensity greater than, or equal to the weak positive control. Specimens reacting with a single antigen band were classified as indeterminate and specimens producing no reactive bands or bands with an intensity less than the weak positive control were classified as negative. HCV RNA Detection. HCV RNA was assayed on plasma or serum samples using the HCV Monitor (v2.0) RT- PCR kit on the COBAS AMPLICOR system (Roche Diagnostics, Switzerland). Data Storage. Demographic, clinical and laboratory data of all subjects whose samples tested positive for anti-HCV by ELISA were kept in a Microsoft Access database for retrieval and analysis. R E S U L T S Of the approximately 55,000 serum samples that were screened for anti-HCV antibodies from 99 to 200, 30,02 were from blood donors. The annual anti-HCV positivity rate among blood donors varied from .4% L A B O R AT O R Y A N A LY S I S O F V I R A L H E PAT I T I S C Table 2. Prevalence of antibodies to hepatitis C virus among blood donors Year No. tested No. positive % positive 1991* 668 6 0.9 1992 2596 34 1.3 1993 3584 51 1.4 1994 2033 23 1.1 1995 2330 20 0.86 1996 2637 25 0.95 1997 2638 26 0.99 1998 2817 20 0.71 1999 2996 19 0.63 2000 3290 23 0.7 2001 4423 25 0.57 *First, second and third generation anti-HCV ELISAs were used to screen blood donors in 1991, 1992–1996, and 1997 –2001, respectively. Figure 2. Identity and location of HCV antigens on a nitrocellulose strip of the recombinant immunoblot assay 8 in 993 to 0.57% in 200, and the mean rate was 0.9% [Table 2]. On average, proportionately more samples tested positive in second generation ELISA than in third generation. Of a total of 2,039 sera that were reactive by ELISA from 994 to 200, ,457 (7.5%) were tested by RIBA [Table 3]. The proportion of samples confirmed posi- tive for anti-HCV antibodies varied widely. It was high among intravenous drug users (95.5%) and patients with liver disease (80.9%), intermediate in patients with hae- moglobinopathies, and low among blood donors (46.5%) [Table 3]. The majority of patients with liver disease had hepatitis, liver cirrhosis or hepatocellular carcinoma. The impact of changing from second to third generation assays is summarized in table 4. Although third generation RIBA confirmed more positive sam- ples than second generation RIBA, it was also associated with slightly more indeterminate results. Plasma samples from 24 ELISA positive subjects were assayed by HCV RT-PCR. These samples were collected in 2000 and 200. Overall HCV RNA was detected in (49.8 %) of the subjects. When results were analysed according to RIBA-3 status, 67.4%, 5.7% and 0% of the RIBA positive, inderterminate and negative sub- jects respectively had HCV RNA [Table 5]. D I S C U S S I O N The introduction of anti-HCV ELISA to screen blood donors has led to a dramatic reduction in post-transfu- sion non-A non-B hepatitis, and the detection of anti- bodies to HCV has become the most practical means of diagnosing infection.5 Over a period of  years, three generations of ELISA were used at SQUH to screen healthy subjects, particularly blood donors, and patients for anti-HCV and two generations of RIBA as supple- mentary test. Based on ELISA, the prevalence of HCV among blood donors in Oman is close to %. However, since only about half of these were confirmed by RIBA, the true prevalence is about 0.5%. On the basis of studies among blood donors that used both ELISA and supplemen- tal testing the lowest anti-HCV prevalence rates (0.0– D H A H R Y E T A L Table 3. Confirmatory assay of anti-HCV ELISA positive samples from blood donors and patients. Clinical category No. samples ELISA positive No. tested by RIBA % RIBA positive % RIBA indeterminate % RIBA negative Liver disease 406 340 80.9 13.8 5.3 Thalassaemia 840 628 69.6 19.8 10.7 Sickle cell disease 126 111 71.2 17.1 11.7 Intra-venous drug abuse 25 22 95.5 4.5 0 Other diseases 133 107 61.7 27.1 11.2 Disease not specified 237 105 60.9 25.7 13.3 Blood donors 272 144 46.5 23.6 29.9 Total 2039 1457 69.3 19.3 11.5 Table 4. Comparison of detection rates of anti-HCV antibodies by second and third generation serological assays Generation ELISA and system % RIBA-2 % RIBA-3 Positive Indeterminate Negative Positive Indeterminate Negative ELISA-3 Quantum (n=117) 61.5 12.8 25.6 –* – – ELISA-2 IMX(n=222) 59.8 21.0 19.2 – – – ELISA-2 AxSYM(n=249) 54.2 13.3 32.5 – – – ELISA-3 AxSYM(n=869) –* – – 69.9 20.8 9.3 *ELISA-positive samples were re-tested by either second, or third generation RIBA. None of the samples was tested by both RIBA-2 and RIBA-3. 9 0.%) have been reported from the United Kingdom9 and Scandinavia.10 Low but slightly higher rates (0.2– 0.5%) have been reported from Western Europe, North America, most areas of Central and South America, and limited regions of Africa.2 The rate reported here for Oman falls into this group. Intermediate rates of anti-HCV prevalence (–5%) have been found in other countries, including neighbouring Yemen11 and Saudi Arabia.12 The proportion of anti-HCV ELISA positive samples that were confirmed by either RIBA-2 or RIBA-3 varied from 47% in blood donors to 80% in patients with a clini- cal diagnosis of hepatitis. These findings are consistent with reports by other investigators.13–15 In populations at low risk for hepatitis C, such as blood donors, the pro- portion of samples confirmed positive by RIBA is low, and varies from 7% to 37%.9,13,14 On the other hand, most (80%–90%) of ELISA-2 or ELISA-3 positive patients with chronic liver disease are RIBA positive.13,15–17 The introduction of third generation RIBA was reported to have resolved many of the indeterminate samples of RIBA-2.14,18,19 In our experience, this was not the case. The proportion of indeterminate samples remained at about 20% when the laboratory changed from second to third generation assays. The results presented here show that HCV RNA was detectable in 67% of RIBA-positive samples, in 6% of RIBA-indeterminates and in none of the samples that were RIBA negative. The mean detection rate was close to 50%. Although our data is based on a small number of subjects, it is comparable to data from larger stud- ies involving blood donors and individuals with normal alanine aminotransferase (ALT) levels.7 In these sub- jects, the percentage that have detectable HCV RNA in serum when tested by PCR assay varies from 70% to 80% for those who are RIBA positive to 2% to 40% for those who are RIBA indeterminate, to none among those who are RIBA negative, giving an overall detection rate of 35% to 45%. Three types of assays (ELISA, RIBA and PCR) were used at SQUH to test for HCV infection. Do diagnostic laboratories need to use all three? Different diagnostic algorithms have been proposed which reflects different opinions on this subject.5–7 There can be no question on the utility of ELISA as a screening test for all subjects. The need for and the choice of supplementary and con- firmatory tests depend on the clinical setting and the likelihood of a true-positive ELISA result. In general, qualitative PCR assay for serum or plasma HCV RNA is the best confirmatory assay and should be used in ELISA positive patients who present with chronic liver disease. There is no need for doing RIBA in such cases. However, ELISA-positive blood donors and individuals with nor- mal ALT levels may be evaluated by RIBA first, PCR for HCV RNA being performed only on those who are RIBA positive or indeterminate. C O N C L U S I O N S At SQUH, we have used ELISA and RIBA to detect anti- bodies to HCV as means of diagnosing hepatitis C. These two tests are relatively easy to perform. 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Increased detection of antibody to hepatitis C virus in renal transplant patients by third generation assays. Amer J Kid Dis, 996, 28, 437–440. D H A H R Y E T A L Laboratory diagnosis of viral hepatitis CThe Sultan Qaboos University Hospital experience *Said H S Al Dhahry1, Jameel C Nograles2, Situsekara M W W B Rajapakse2, Fadhila S S Al Toqi2, Geraldine Z Kaminski2 Materials and Methods Results Discussion Conclusions Acknowledgement References