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The accuracy of SM-HCV rapid test for the detection of antibody to hepatitis C virus
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2001 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 96, No. 3, 2001 ISSN 0002-9270/01/$20.00 PII S0002-9270(00)02421-7
The Accuracy of SM-HCV Rapid Test for the Detection of Antibody to Hepatitis C Virus Man-Fung Yuen, M.B.B.S., Chee-Kin Hui, M.B.B.S., John Chi-Hang Yuen, B.Sc., John Lap-Ping Young, B.Sc., and Ching-Lung Lai, M.D. Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
OBJECTIVE: Conventional tests for antibody to hepatitis C virus (HCV) require considerable time before results are available. The aim of this study is to examine the accuracy of a new quick test (SM-HCV Rapid Test) for the detection of antibody to hepatitis C virus with reference to the wellestablished third generation enzyme immunoblot assay (EIA-3; Abbott Laboratories, Chicago, IL). METHODS: A total of 290 subjects (100 patients with chronic hepatitis C infections, 95 patients with other chronic liver diseases, 95 healthy subjects) were recruited. Thirty microliters of serum was tested for anti-HCV by SM-HCV Rapid Test according to the manufacturer’s instruction. Liver function tests and serum HCV RNA by polymerase chain reaction (PCR) were measured. RESULTS: In the 100 patients positive for anti-HCV by EIA-3, 98 of these patients were also positive for anti-HCV by SM-HCV Rapid Test. In the 95 patients with other chronic liver diseases, 94 samples were negative for antiHCV by both EIA-3 and SM-HCV Rapid Test. The remaining one patient was positive for anti-HCV by the EIA-3 but negative by the SM-HCV Rapid Test. In the 95 controls, which were negative for anti-HCV by EIA-3, all were also negative for anti-HCV by SM-HCV Rapid Test and HCV RNA by PCR. Using EIA-3 as the gold standard screening test for anti-HCV, the sensitivity and the specificity of SM-HCV Rapid Test were 98% and 100%, respectively. The positive predictive value and negative predictive value of SM-HCV Rapid Test were 100% and 97.9%, respectively. CONCLUSIONS: SM-HCV Rapid Test is a reliable test with high sensitivity and specificity. The anti-HCV result can be available within a very short period of time. It is a useful screening test for anti-HCV. (Am J Gastroenterol 2001;96: 838 – 841. © 2001 by Am. Coll. of Gastroenterology)
INTRODUCTION After the discovery of hepatitis C virus (HCV) in 1989, there is continuous modification of various assays to refine the accuracy for detecting the serological marker for HCV infection, that is, antibody to HCV (anti-HCV). The main
screening tests for anti-HCV include enzyme immunoassays (EIA) by Abbott Laboratories (Chicago, IL) and by Ortho Diagnostic Systems (Raritan, NJ) (1–3). The third generation of EIA (EIA-3) has reached a high sensitivity of 97% (2). Confirmatory anti-HCV testing by recombinant immunoblot assay is no longer necessary, particularly in high-risk patients (2, 4). The common limitation for the serological assays is the time required to perform the assay. It takes approximately 3 h to finish the EIA-3. In addition, to minimize the cost of running an assay, laboratories usually perform the tests when an adequate number of serum samples have been collected. Therefore, it may take a considerable time before the results are available. This problem is particularly important in centers with a relatively small population of high-risk individuals. Rapid test for testing anti-HCV may ameliorate this problem and can be used as a tool for screening subjects. It is particularly useful in some conditions in which urgent results are required, such as patients requiring urgent hemodialysis with unknown hepatitis C status. However, before implementing a rapid test as a screening tool, it should be verified with a standard test with regard to sensitivity and specificity. This study aims at comparing the anti-HCV results of a rapid test, SM-HCV Rapid Test (SERO-Med Laborspezialita¨ten GmbH, Eichsta¨tt, Germany) and the standard EIA-3.
PATIENTS AND METHODS A total of 290 subjects were recruited. The median age was 37.5 yr (12–78 yr) with male-to-female ratio of 185:105. One hundred ninety-five patients were followed up in the Hepatitis Clinic, The University of Hong Kong, Hong Kong. Of these, 100 patients were anti-HCV positive by the EIA-3 (Abbott Laboratories). Another 95 patients (75 patients were hepatitis B surface antigen positive, 14 patients had autoimmune hepatitis, four patients had fatty liver, and two patients had alcoholic liver disease) without prior testing of anti-HCV were also tested for anti-HCV by EIA-3 as well as SM-HCV Rapid Test (in both the whole blood and the serum). The remaining 95 subjects were healthy controls negative for anti-HCV by the EIA-3. The rapid test for anti-HCV was performed using the
AJG – March, 2001
Accuracy of SM-HCV Rapid Test for Anti-HCV
839
Figure 1. Diagrammatic representation of the methodology according to the instructions given by SM-HCV Rapid Test (SERO-Med Laborspezialita¨ten GmbH, Eichsta¨tt, Germany).
SM-HCV Rapid Test, which was a single use device system. The test was based on the principle of sandwich enzyme immunoassay for the semiquantitative assessment of HCV antibody in serum or whole blood. The active ingredients of
the reaction unit were monoclonal anticolloidal gold antibodies (control bar) and purified recombinant hepatitis C, NS3, NS4, and core antigens (dot). The test performance is illustrated in Figure 1. Briefly, 30 – 40 l of serum or whole
840
Yuen et al.
AJG – Vol. 96, No. 3, 2001
Table 1. Results of Anti-HCV Antibody Testing by Two Different Assays SM-HCV Rapid Test
Enzyme Immunoassay-3
Positive
Negative
Positive
Negative
98 0 0
2 95 95
100 1 0
0 94 95
Chronic hepatitis C patients (n ⫽ 100) Patients with other chronic liver diseases (n ⫽ 95) Control (n ⫽ 95)
blood was taken through a plastic capillary. The sample was then put to the reaction well, which already contained 10 drops of gold conjugate solution (mouse monoclonal antihuman IgG conjugated with gold particles). All the solution was transferred from the reaction well to a prefilter well and allowed to soak through the prefilter. Another 10 drops of gold conjugated solution were added to the prefilter, which was then removed after soaking. The results were read. Result was regarded as positive if a large round spot was visible together with the presence of a dot bar, which served as internal control. Strong positive meant the round spot was darker than all spots in the dot bar. Weak positive meant the round spot was darker than the center dot in the dot bar but less defined than the other two dots. If only the dot bar was visible, the result was negative. Result was considered inconclusive if only the large round spot was visible or both the spot and dot bar were not visible. The whole test took about 3 min. All samples were tested in duplicate. To examine whether the test was as accurate for the whole blood and for the serum, both the whole blood and the serum of the 95 patients with chronic liver diseases were tested. For those with anti-HCV positive by EIA-3, the liver function tests were taken. The HCV RNA was checked by qualitative in-house nested polymerase chain reaction (PCR) assay in all blood samples. The method for the PCR assay has been described previously (5).
RESULTS In the 100 patients positive for anti-HCV by EIA-3, 73 (73%) patients were HCV RNA positive by PCR. Fifty-five of 93 (59.1%) patients had abnormal ALT levels. Ninetyeight of these patients were also positive for anti-HCV by SM-HCV Rapid Test. There was no discordance of the results in the duplicate tests performed on the samples. In the 95 patients with other chronic liver diseases without prior testing for anti-HCV, 94 were negative for anti-HCV by both EIA-3 and SM-HCV Rapid Test. Only one patient with chronic hepatitis B infection, who was positive for anti-HCV by EIA-3, was negative by the SM-HCV Rapid Test. There was no discrepancy of the results by SM-HCV Rapid Test when the tests were performed on whole blood and serum. In the 95 control sera negative for anti-HCV by EIA-3, all were also negative for anti-HCV by SM-HCV Rapid Test and HCV RNA by PCR. The results are listed in Table 1. Using EIA-3 as the gold standard screening test for antiHCV, the sensitivity and the specificity of SM-HCV Rapid
Test were 98% and 100%, respectively. The positive predictive value and negative predictive value of SM-HCV Rapid Test were 100% and 97.9%, respectively. For the two patients who were anti-HCV positive by EIA but negative by SM-HCV Rapid Test, the HCV RNA were both negative by PCR. The anti-HCV positivity had been confirmed previously by recombinant immunoblot assay 3.0 (Chiron Corporation, Emeryville, CA). The ALT levels were normal in both patients.
DISCUSSION This study showed that the SM-HCV Rapid Test was a reliable test for screening anti-HCV in the population. The results of SM-HCV Rapid Test correlated very well with the standard EIA-3 test. It had excellent sensitivity and specificity. SM-HCV Rapid Test has several advantages compared to the conventional tests for anti-HCV. First, it requires only a very small amount of blood or serum. Retrospective anti-HCV testing can be performed even when only a very small amount of stored serum is left and the patient does not need to have repeated venipuncture. Second, the result of anti-HCV testing can be available in a very short time (around 3 min) compared to the longer time requirement for EIA-3 (around 3 h). It is particularly important in situations where urgent anti-HCV result is required, such as when a patient requires urgent hemodialysis, and also in an outpatient department. Furthermore, to minimize the cost of running EIA-3, laboratories usually perform the test only if adequate test samples are collected. This will further delay the availability of the anti-HCV result. In contrast, SM-HCV Rapid Test can be performed on an individual sample basis. By checking the blood of the patients with SM-HCV Rapid Test, the psychological stress can be quickly relieved in members of the medical professional who have accidental needle prick injuries. Third, SM-HCV Rapid Test does not require technical expertise. This is a significant benefit in centers where technical expertise is lacking or 24-h viral laboratory services are not available. In this study, two patients were anti-HCV positive by EIA-3 but negative by SM-HCV Rapid Test. They were HCV RNA negative with normal ALT levels. In self-limited hepatitis C infection, the anti-HCV antibody progressively decreases, although it may be detectable by sensitive assay (6). These two patients might have had acute hepatitis C infections that had resolved and in whom the anti-HCV titers
AJG – March, 2001
had decreased to such low levels that they could only be detected by the EIA-3 but not by the SM-HCV Rapid Test. In conclusion, SM-HCV Rapid Test is a reliable test with high sensitivity and specificity. The anti-HCV result can be available within a very short period of time. It is a useful screening test for anti-HCV.
ACKNOWLEDGMENT We thank Dr. Sai-Kit Tang and Dr. Kam-Suen Lee for the collection of the data in this study. This study was sponsored by Schering-Pough Corporation, Kenilworth, NJ. Reprint requests and correspondence: Prof. Ching-Lung Lai, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. Received Mar. 23, 2000; accepted Sep. 20, 2000.
Accuracy of SM-HCV Rapid Test for Anti-HCV
841
REFERENCES 1. Younossi Z, McHutchison J. Serological tests for HCV infection. Viral Hepatitis Rev 1996;2:161–73. 2. Gretch DR. Diagnostic tests for hepatitis C. Hepatology 1997; 26(3 suppl 1):43S-47S. 3. Bonanni P, Icardi GC, Raffo AM, et al. Analytical and laboratory evaluation of a new fully-automated third generation enzyme immunoassay for the detection of antibodies to the hepatitis C virus. J Virol Methods 1996;62:113–22. 4. Krarup HB, Jacobsen SE, Varming K, et al. Performance of hepatitis C (HCV) antibody test systems in relation to HCVRNA detection in the diagnosis of HCV infection. Dan Med Bell 1998;45:89 –91. 5. Lin HJ, Hollinger FB, Mizokami M, et al. A polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pairs. J Med Virol 1992;3:220 –5. 6. Marcellin P. Hepatitis C: The clinical spectrum of the disease. J Hepatol 1999;31(suppl 1):17–24.
Vol. 96, No. 3, 2001 ISSN 0002-9270/01/$20.00 PII S0002-9270(00)02421-7
The Accuracy of SM-HCV Rapid Test for the Detection of Antibody to Hepatitis C Virus Man-Fung Yuen, M.B.B.S., Chee-Kin Hui, M.B.B.S., John Chi-Hang Yuen, B.Sc., John Lap-Ping Young, B.Sc., and Ching-Lung Lai, M.D. Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
OBJECTIVE: Conventional tests for antibody to hepatitis C virus (HCV) require considerable time before results are available. The aim of this study is to examine the accuracy of a new quick test (SM-HCV Rapid Test) for the detection of antibody to hepatitis C virus with reference to the wellestablished third generation enzyme immunoblot assay (EIA-3; Abbott Laboratories, Chicago, IL). METHODS: A total of 290 subjects (100 patients with chronic hepatitis C infections, 95 patients with other chronic liver diseases, 95 healthy subjects) were recruited. Thirty microliters of serum was tested for anti-HCV by SM-HCV Rapid Test according to the manufacturer’s instruction. Liver function tests and serum HCV RNA by polymerase chain reaction (PCR) were measured. RESULTS: In the 100 patients positive for anti-HCV by EIA-3, 98 of these patients were also positive for anti-HCV by SM-HCV Rapid Test. In the 95 patients with other chronic liver diseases, 94 samples were negative for antiHCV by both EIA-3 and SM-HCV Rapid Test. The remaining one patient was positive for anti-HCV by the EIA-3 but negative by the SM-HCV Rapid Test. In the 95 controls, which were negative for anti-HCV by EIA-3, all were also negative for anti-HCV by SM-HCV Rapid Test and HCV RNA by PCR. Using EIA-3 as the gold standard screening test for anti-HCV, the sensitivity and the specificity of SM-HCV Rapid Test were 98% and 100%, respectively. The positive predictive value and negative predictive value of SM-HCV Rapid Test were 100% and 97.9%, respectively. CONCLUSIONS: SM-HCV Rapid Test is a reliable test with high sensitivity and specificity. The anti-HCV result can be available within a very short period of time. It is a useful screening test for anti-HCV. (Am J Gastroenterol 2001;96: 838 – 841. © 2001 by Am. Coll. of Gastroenterology)
INTRODUCTION After the discovery of hepatitis C virus (HCV) in 1989, there is continuous modification of various assays to refine the accuracy for detecting the serological marker for HCV infection, that is, antibody to HCV (anti-HCV). The main
screening tests for anti-HCV include enzyme immunoassays (EIA) by Abbott Laboratories (Chicago, IL) and by Ortho Diagnostic Systems (Raritan, NJ) (1–3). The third generation of EIA (EIA-3) has reached a high sensitivity of 97% (2). Confirmatory anti-HCV testing by recombinant immunoblot assay is no longer necessary, particularly in high-risk patients (2, 4). The common limitation for the serological assays is the time required to perform the assay. It takes approximately 3 h to finish the EIA-3. In addition, to minimize the cost of running an assay, laboratories usually perform the tests when an adequate number of serum samples have been collected. Therefore, it may take a considerable time before the results are available. This problem is particularly important in centers with a relatively small population of high-risk individuals. Rapid test for testing anti-HCV may ameliorate this problem and can be used as a tool for screening subjects. It is particularly useful in some conditions in which urgent results are required, such as patients requiring urgent hemodialysis with unknown hepatitis C status. However, before implementing a rapid test as a screening tool, it should be verified with a standard test with regard to sensitivity and specificity. This study aims at comparing the anti-HCV results of a rapid test, SM-HCV Rapid Test (SERO-Med Laborspezialita¨ten GmbH, Eichsta¨tt, Germany) and the standard EIA-3.
PATIENTS AND METHODS A total of 290 subjects were recruited. The median age was 37.5 yr (12–78 yr) with male-to-female ratio of 185:105. One hundred ninety-five patients were followed up in the Hepatitis Clinic, The University of Hong Kong, Hong Kong. Of these, 100 patients were anti-HCV positive by the EIA-3 (Abbott Laboratories). Another 95 patients (75 patients were hepatitis B surface antigen positive, 14 patients had autoimmune hepatitis, four patients had fatty liver, and two patients had alcoholic liver disease) without prior testing of anti-HCV were also tested for anti-HCV by EIA-3 as well as SM-HCV Rapid Test (in both the whole blood and the serum). The remaining 95 subjects were healthy controls negative for anti-HCV by the EIA-3. The rapid test for anti-HCV was performed using the
AJG – March, 2001
Accuracy of SM-HCV Rapid Test for Anti-HCV
839
Figure 1. Diagrammatic representation of the methodology according to the instructions given by SM-HCV Rapid Test (SERO-Med Laborspezialita¨ten GmbH, Eichsta¨tt, Germany).
SM-HCV Rapid Test, which was a single use device system. The test was based on the principle of sandwich enzyme immunoassay for the semiquantitative assessment of HCV antibody in serum or whole blood. The active ingredients of
the reaction unit were monoclonal anticolloidal gold antibodies (control bar) and purified recombinant hepatitis C, NS3, NS4, and core antigens (dot). The test performance is illustrated in Figure 1. Briefly, 30 – 40 l of serum or whole
840
Yuen et al.
AJG – Vol. 96, No. 3, 2001
Table 1. Results of Anti-HCV Antibody Testing by Two Different Assays SM-HCV Rapid Test
Enzyme Immunoassay-3
Positive
Negative
Positive
Negative
98 0 0
2 95 95
100 1 0
0 94 95
Chronic hepatitis C patients (n ⫽ 100) Patients with other chronic liver diseases (n ⫽ 95) Control (n ⫽ 95)
blood was taken through a plastic capillary. The sample was then put to the reaction well, which already contained 10 drops of gold conjugate solution (mouse monoclonal antihuman IgG conjugated with gold particles). All the solution was transferred from the reaction well to a prefilter well and allowed to soak through the prefilter. Another 10 drops of gold conjugated solution were added to the prefilter, which was then removed after soaking. The results were read. Result was regarded as positive if a large round spot was visible together with the presence of a dot bar, which served as internal control. Strong positive meant the round spot was darker than all spots in the dot bar. Weak positive meant the round spot was darker than the center dot in the dot bar but less defined than the other two dots. If only the dot bar was visible, the result was negative. Result was considered inconclusive if only the large round spot was visible or both the spot and dot bar were not visible. The whole test took about 3 min. All samples were tested in duplicate. To examine whether the test was as accurate for the whole blood and for the serum, both the whole blood and the serum of the 95 patients with chronic liver diseases were tested. For those with anti-HCV positive by EIA-3, the liver function tests were taken. The HCV RNA was checked by qualitative in-house nested polymerase chain reaction (PCR) assay in all blood samples. The method for the PCR assay has been described previously (5).
RESULTS In the 100 patients positive for anti-HCV by EIA-3, 73 (73%) patients were HCV RNA positive by PCR. Fifty-five of 93 (59.1%) patients had abnormal ALT levels. Ninetyeight of these patients were also positive for anti-HCV by SM-HCV Rapid Test. There was no discordance of the results in the duplicate tests performed on the samples. In the 95 patients with other chronic liver diseases without prior testing for anti-HCV, 94 were negative for anti-HCV by both EIA-3 and SM-HCV Rapid Test. Only one patient with chronic hepatitis B infection, who was positive for anti-HCV by EIA-3, was negative by the SM-HCV Rapid Test. There was no discrepancy of the results by SM-HCV Rapid Test when the tests were performed on whole blood and serum. In the 95 control sera negative for anti-HCV by EIA-3, all were also negative for anti-HCV by SM-HCV Rapid Test and HCV RNA by PCR. The results are listed in Table 1. Using EIA-3 as the gold standard screening test for antiHCV, the sensitivity and the specificity of SM-HCV Rapid
Test were 98% and 100%, respectively. The positive predictive value and negative predictive value of SM-HCV Rapid Test were 100% and 97.9%, respectively. For the two patients who were anti-HCV positive by EIA but negative by SM-HCV Rapid Test, the HCV RNA were both negative by PCR. The anti-HCV positivity had been confirmed previously by recombinant immunoblot assay 3.0 (Chiron Corporation, Emeryville, CA). The ALT levels were normal in both patients.
DISCUSSION This study showed that the SM-HCV Rapid Test was a reliable test for screening anti-HCV in the population. The results of SM-HCV Rapid Test correlated very well with the standard EIA-3 test. It had excellent sensitivity and specificity. SM-HCV Rapid Test has several advantages compared to the conventional tests for anti-HCV. First, it requires only a very small amount of blood or serum. Retrospective anti-HCV testing can be performed even when only a very small amount of stored serum is left and the patient does not need to have repeated venipuncture. Second, the result of anti-HCV testing can be available in a very short time (around 3 min) compared to the longer time requirement for EIA-3 (around 3 h). It is particularly important in situations where urgent anti-HCV result is required, such as when a patient requires urgent hemodialysis, and also in an outpatient department. Furthermore, to minimize the cost of running EIA-3, laboratories usually perform the test only if adequate test samples are collected. This will further delay the availability of the anti-HCV result. In contrast, SM-HCV Rapid Test can be performed on an individual sample basis. By checking the blood of the patients with SM-HCV Rapid Test, the psychological stress can be quickly relieved in members of the medical professional who have accidental needle prick injuries. Third, SM-HCV Rapid Test does not require technical expertise. This is a significant benefit in centers where technical expertise is lacking or 24-h viral laboratory services are not available. In this study, two patients were anti-HCV positive by EIA-3 but negative by SM-HCV Rapid Test. They were HCV RNA negative with normal ALT levels. In self-limited hepatitis C infection, the anti-HCV antibody progressively decreases, although it may be detectable by sensitive assay (6). These two patients might have had acute hepatitis C infections that had resolved and in whom the anti-HCV titers
AJG – March, 2001
had decreased to such low levels that they could only be detected by the EIA-3 but not by the SM-HCV Rapid Test. In conclusion, SM-HCV Rapid Test is a reliable test with high sensitivity and specificity. The anti-HCV result can be available within a very short period of time. It is a useful screening test for anti-HCV.
ACKNOWLEDGMENT We thank Dr. Sai-Kit Tang and Dr. Kam-Suen Lee for the collection of the data in this study. This study was sponsored by Schering-Pough Corporation, Kenilworth, NJ. Reprint requests and correspondence: Prof. Ching-Lung Lai, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. Received Mar. 23, 2000; accepted Sep. 20, 2000.
Accuracy of SM-HCV Rapid Test for Anti-HCV
841
REFERENCES 1. Younossi Z, McHutchison J. Serological tests for HCV infection. Viral Hepatitis Rev 1996;2:161–73. 2. Gretch DR. Diagnostic tests for hepatitis C. Hepatology 1997; 26(3 suppl 1):43S-47S. 3. Bonanni P, Icardi GC, Raffo AM, et al. Analytical and laboratory evaluation of a new fully-automated third generation enzyme immunoassay for the detection of antibodies to the hepatitis C virus. J Virol Methods 1996;62:113–22. 4. Krarup HB, Jacobsen SE, Varming K, et al. Performance of hepatitis C (HCV) antibody test systems in relation to HCVRNA detection in the diagnosis of HCV infection. Dan Med Bell 1998;45:89 –91. 5. Lin HJ, Hollinger FB, Mizokami M, et al. A polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pairs. J Med Virol 1992;3:220 –5. 6. Marcellin P. Hepatitis C: The clinical spectrum of the disease. J Hepatol 1999;31(suppl 1):17–24.