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A rapid test for the visual detection of anti-hepatitis C virus antibodies in whole blood
Clinica Chimica Acta 288 (1999) 91–96 www.elsevier.com / locate / clinchim
A rapid test for the visual detection of anti-hepatitis C virus antibodies in whole blood a,
a
b
Lucio Montebugnoli *, Giorgio Borea , Rita Miniero , Giuseppe Sprovieri b b
a Department of Dentistry, University of Bologna, Bologna, Italy Department of Clinical Pathology, S. Orsola Hospital, Bologna, Italy
Received 15 March 1999; received in revised form 19 July 1999; accepted 20 July 1999
Abstract A rapid test for the visual detection of anti-HCV antibodies in whole blood was evaluated in its accuracy when compared with EIA method. The rapid test was performed blind on 50 HCV EIA-positive (adsorbance greater than 3.0) and on 50 HCV EIA-negative samples. Each whole blood sample was 1:20, 1:50 and 1:100 diluted with saline solution for a total of 400 samples. Results showed a sensitivity of 100% when whole blood was tested, 96% when 1:20 diluted blood was tested, 30% when 1:50 diluted blood was tested and 4% when 1:100 diluted blood was tested. The specificity gave also better results and only one false-positive was found in all samples tested. The test took less than 3 min and only a mechanical pipette was required. In conclusion, the HCV Ab rapid test showed a very high accuracy and could be very useful for the detection of HCV-positive subjects in situations where rapid results are required or technical expertise is limited. 1999 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C; Laboratory test; Cross-infection
1. Introduction The enzyme immunoassay (EIA) method is the most frequently used to detect antibodies to hepatitis C virus (HCV). While EIAs are highly accurate and *Corresponding author. Tel.: 1 39-51-278-011; fax: 1 39-51-225-208. E-mail address: [email protected] (L. Montebugnoli) 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00146-1
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L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
applicable in many situations, there is the need today for alternatives to be applied in some instances where rapid results are required or where capabilities are less than optimal [1]. To the best of our knowledge there have been few reports on the use of rapid HCV assays and all were performed only on fresh serum samples [2–4]. Recently, an anti-HCV Ab rapid test has been proposed as a rapid and simple test applicable directly on the whole blood, requiring no laboratory equipment and producing results in less than 3 min. The aim of the present research is to evaluate accuracy and performance characteristics of the method tested blind directly on the whole blood of two paired groups of 50 HCV EIA antibody-positive samples and 50 HCV EIA antibody-negative samples.
2. Materials and methods A total of 100 whole blood samples that had been collected routinely as a part of a normal service of the S. Orsola Central laboratory in Bologna were used in the study. Fifty whole bloods were obtained from HCV EIA antibody-negative and 50 from HCV EIA antibody-positive samples (ORTHO HCV 3.0 ELISA Chiron Corporation, confirmed by RIBA TEST 3.0 Chiron Corporation). All positive serum samples had adsorbances greater than 3.0. In addition, each whole blood sample was 1:20, 1:50, 1:100 diluted with saline solution for a total of 400 samples (100 samples of whole blood, 100 samples of 1:20 diluted blood, 100 samples of 1:50 diluted blood and 100 samples of 1:100 diluted blood). All samples were tested blind using the rapid test within 24 h of collection and the results were compared with those obtained with the EIA test. The anti-HCV Ab rapid test (standardized to WHO 1st IRP 75 / 537 and distributed by Thema Ricerca) is a rapid direct binding test for the visual detection of anti-hepatitis C antibody (anti-HCV Ab) in whole blood. The results are read visually without any instrument. HCV test is based on the principle of sandwich enzyme immunoassay for the semiquantitative determination of anti-HCV Ab in whole blood. Synthetic and recombinant HCV antigens were employed to specifically identify anti-HCV Ab. The entire test takes only about 3 min and does not require to be timed since the absorption procedure is preprogrammed for optimal performance. The results can be stored as a permanent record.
2.1. Description of the test device The test device is a plastic manufactured article with an internal chamber
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
93
where the entire procedure takes place and an external reaction well where the mixture of sample and conjugate solution is formed. An adsorbent membrane (filter) containing recombinant HCV antigens is encased inside the plastic device, subdividing the internal chamber into two different compartments. The access to the compartment above the filter is regulated by a further adsorbent membrane (pre-filter) which allows the mixture to soak through very slowly and permits a gentle and prolonged contact of the mixture with the filter membrane. The compartment below the filter operates as a reservoir collecting the mixture soaking through the filters at the end of each procedure.
2.1.1. Principles of the test procedure In the test procedure the blood sample is initially mixed with a solution containing gold-conjugated anti-human antibodies. If anti-HCV antibodies are present in the blood sample, immunocomplexes are formed. The reaction mixture is then allowed to reach the filter membrane by slowly soaking through the pre-filter membrane. As the reaction mixture continues to flow along the test membrane the complexes bind to the HCV antigens adsorbed on the test zone of the membrane and produce a dark rounded spot. Unbound conjugate binds to the reagents immobilized in the control zone of the membrane filter producing a dot bar, demonstrating proper performance of the test. 2.2. Test procedure All reagents, test devices, specimens, reference and other materials were brought to room temperature prior to testing. 1. Four drops of enzyme conjugate solution are added to the reaction well of the test device. 2. A drop of blood sample is added to the reaction well and mixed with enzyme conjugate solution by pipetting the mixture up and down. 3. All the mixture is then transferred from the reaction well and added to the prefilter well. A further 10 drops of enzyme conjugate solution are added and the specimen is allowed to completely soak through the prefilter. 4. After about 3 min the mixture is completely soaked through the filters and the prefilter is removed and discarded. 5. Results are read directly on the filter and interpreted as follows: • inconclusive: only a round spot (.) is visible; test has to be repeated; • negative: only a dot bar ( . . . ) is visible; and • positive: both a round spot (.) and a dot bar ( . . . ) are visible.
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L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
Table 1 Number of positive and negative results obtained with the rapid test in EIA positive whole and diluted blood samples HCV-positive EIA test
Positive Negative Sensitivity (%)
Whole
Diluted blood
blood
1:20
1:50
1:100
50 0
48 2
15 35
2 48
100
96
30
4
3. Results Results observed in whole and diluted blood are showed in Tables 1 and 2 and refer to the accuracy of the test with respect to the EIA test which was the reference method. Results give evidence of a good sensitivity (expressed by the percentage of positive results in HCV EIA-positive samples) of the rapid test when compared with EIA test up to a 1:20 dilution of the whole blood sample. The specificity (expressed by the percentage of negative results in HCV EIA-negative samples) of the rapid test also gave better results with respect to the sensitivity. Only one false-positive was found in all samples tested.
4. Discussion The diagnosis of HCV infections is most frequently accomplished using EIA methods. The methods are highly accurate but they require sophisticated laboratory equipment and over 2 h for completion. Alternatives to these methods are today available and they can be applied in some particular instances: where Table 2 Number of positive and negative results obtained with the rapid test in EIA negative whole and diluted blood samples HCV-negative EIA test Whole
Diluted blood
blood
1:20
1:50
1:100
Positive Negative
1 49
0 50
0 50
0 50
Specificity (%)
98
100
100
100
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
95
rapid results are required (emergency transfusions and transplantation services, or at the time of an accidental percutaneous inoculation involving a health care worker), where facilities and capabilities are less than optimal (small blood centers or in case of epidemiological studies) or, as a complementary aid, in physician’s offices and dental surgeries in order to reduce the risk of patient-topatient transmission [5–8]. The results of the present research show that the rapid HCV assay can be easily and effectively performed for this purpose. The test is extremely easy to carry out and interpret and a quality control measure ensures that it is performed properly. The results are produced in less than 3 min and only a mechanical pipette is required. Further, contrary to other rapid tests previously described [2–4], the test can be performed on whole blood, thus avoiding sophisticated procedures in order to separate serum from cells. The high sensitivity expressed by the test up to 1:20 dilution of the blood, ensures optimal results even in the presence of blood contaminants, for example oral fluids, and can allow to formulate a precise diagnosis even from saliva samples, provided that little amounts of blood, during spontaneous or provoked gingival bleeding, are included. Therefore, it can represent a useful method for a rapid identification of HCV-positive subjects just before the onset of a dental treatment allowing the dentist to improve the procedures for cross-infection control. Further, the possibility to perform accurate oral fluid testing for HCV antibody should stimulate epidemiological studies on large population groups where reluctance to consent to venipuncture is an impediment to epidemiological evaluation. In addition to the high sensitivity, the method showed a very high specificity considering that only one false-positive was detected in all samples tested. This property is welcome in order to avoid unjustified alarms in case of HCVnegative subjects.
Acknowledgements This work was supported by Ditta Castellini s.p.a.
References [1] Klein RS, Freeman K, Taylor PE, Stevens CE. Occupational risk for hepatitis C virus infection among New York City dentists. Lancet 1991;338:1539–42. [2] Constantine NT, Callahan JC, Watts DM. Successful use of two rapid HCV assays in a high prevalence Romanian population. J Chin Lab Anal 1994;8:332–4.
96
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
[3] Poovorawan Y, Theamboonlers A, Chumdermpadetsuk S, Thong CP. Comparative results in detection of HCV antibodies by using a rapid HCV test, ELISA and immunoblot. Southeast Asian J Trop Med Public Health 1994;25(4):647–9. [4] Mvere D, Constantine NT, Katsawde E, Tobaiwa O, Dambire S, Corcoran P. Rapid and simple hepatitis assay:encouraging results from a blood donor population in Zimbabwe. Bull World Health Organ 1996;74(1):19–24. [5] Chen M, Yun ZB, Sallberg M, Schvarcz R, Bergquist I, Berglund HB. Detection of hepatitis C virus RNA in the cell fraction of saliva before and after oral surgery. J Med Virol 1995;45(2):223–6. [6] Piazza M, Borgia C, Picciotto L, Nappa S, Cicciarello S, Orlando R. Detection of hepatitis C virus-RNA by polymerase chain reaction in dental surgeries. J Med Virol 1995;45(1):40–2. [7] Allander T, Gruber A, Naghavi M. Frequent patient-to-patient transmission of hepatitis C in haematology ward. Lancet 1995;345:603–7. [8] Porter SR, Lodi G. Hepatitis C virus (HCV) — an occupational risk to dentists? Br Dent J 1996;180(12):473–4.
A rapid test for the visual detection of anti-hepatitis C virus antibodies in whole blood a,
a
b
Lucio Montebugnoli *, Giorgio Borea , Rita Miniero , Giuseppe Sprovieri b b
a Department of Dentistry, University of Bologna, Bologna, Italy Department of Clinical Pathology, S. Orsola Hospital, Bologna, Italy
Received 15 March 1999; received in revised form 19 July 1999; accepted 20 July 1999
Abstract A rapid test for the visual detection of anti-HCV antibodies in whole blood was evaluated in its accuracy when compared with EIA method. The rapid test was performed blind on 50 HCV EIA-positive (adsorbance greater than 3.0) and on 50 HCV EIA-negative samples. Each whole blood sample was 1:20, 1:50 and 1:100 diluted with saline solution for a total of 400 samples. Results showed a sensitivity of 100% when whole blood was tested, 96% when 1:20 diluted blood was tested, 30% when 1:50 diluted blood was tested and 4% when 1:100 diluted blood was tested. The specificity gave also better results and only one false-positive was found in all samples tested. The test took less than 3 min and only a mechanical pipette was required. In conclusion, the HCV Ab rapid test showed a very high accuracy and could be very useful for the detection of HCV-positive subjects in situations where rapid results are required or technical expertise is limited. 1999 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C; Laboratory test; Cross-infection
1. Introduction The enzyme immunoassay (EIA) method is the most frequently used to detect antibodies to hepatitis C virus (HCV). While EIAs are highly accurate and *Corresponding author. Tel.: 1 39-51-278-011; fax: 1 39-51-225-208. E-mail address: [email protected] (L. Montebugnoli) 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00146-1
92
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
applicable in many situations, there is the need today for alternatives to be applied in some instances where rapid results are required or where capabilities are less than optimal [1]. To the best of our knowledge there have been few reports on the use of rapid HCV assays and all were performed only on fresh serum samples [2–4]. Recently, an anti-HCV Ab rapid test has been proposed as a rapid and simple test applicable directly on the whole blood, requiring no laboratory equipment and producing results in less than 3 min. The aim of the present research is to evaluate accuracy and performance characteristics of the method tested blind directly on the whole blood of two paired groups of 50 HCV EIA antibody-positive samples and 50 HCV EIA antibody-negative samples.
2. Materials and methods A total of 100 whole blood samples that had been collected routinely as a part of a normal service of the S. Orsola Central laboratory in Bologna were used in the study. Fifty whole bloods were obtained from HCV EIA antibody-negative and 50 from HCV EIA antibody-positive samples (ORTHO HCV 3.0 ELISA Chiron Corporation, confirmed by RIBA TEST 3.0 Chiron Corporation). All positive serum samples had adsorbances greater than 3.0. In addition, each whole blood sample was 1:20, 1:50, 1:100 diluted with saline solution for a total of 400 samples (100 samples of whole blood, 100 samples of 1:20 diluted blood, 100 samples of 1:50 diluted blood and 100 samples of 1:100 diluted blood). All samples were tested blind using the rapid test within 24 h of collection and the results were compared with those obtained with the EIA test. The anti-HCV Ab rapid test (standardized to WHO 1st IRP 75 / 537 and distributed by Thema Ricerca) is a rapid direct binding test for the visual detection of anti-hepatitis C antibody (anti-HCV Ab) in whole blood. The results are read visually without any instrument. HCV test is based on the principle of sandwich enzyme immunoassay for the semiquantitative determination of anti-HCV Ab in whole blood. Synthetic and recombinant HCV antigens were employed to specifically identify anti-HCV Ab. The entire test takes only about 3 min and does not require to be timed since the absorption procedure is preprogrammed for optimal performance. The results can be stored as a permanent record.
2.1. Description of the test device The test device is a plastic manufactured article with an internal chamber
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
93
where the entire procedure takes place and an external reaction well where the mixture of sample and conjugate solution is formed. An adsorbent membrane (filter) containing recombinant HCV antigens is encased inside the plastic device, subdividing the internal chamber into two different compartments. The access to the compartment above the filter is regulated by a further adsorbent membrane (pre-filter) which allows the mixture to soak through very slowly and permits a gentle and prolonged contact of the mixture with the filter membrane. The compartment below the filter operates as a reservoir collecting the mixture soaking through the filters at the end of each procedure.
2.1.1. Principles of the test procedure In the test procedure the blood sample is initially mixed with a solution containing gold-conjugated anti-human antibodies. If anti-HCV antibodies are present in the blood sample, immunocomplexes are formed. The reaction mixture is then allowed to reach the filter membrane by slowly soaking through the pre-filter membrane. As the reaction mixture continues to flow along the test membrane the complexes bind to the HCV antigens adsorbed on the test zone of the membrane and produce a dark rounded spot. Unbound conjugate binds to the reagents immobilized in the control zone of the membrane filter producing a dot bar, demonstrating proper performance of the test. 2.2. Test procedure All reagents, test devices, specimens, reference and other materials were brought to room temperature prior to testing. 1. Four drops of enzyme conjugate solution are added to the reaction well of the test device. 2. A drop of blood sample is added to the reaction well and mixed with enzyme conjugate solution by pipetting the mixture up and down. 3. All the mixture is then transferred from the reaction well and added to the prefilter well. A further 10 drops of enzyme conjugate solution are added and the specimen is allowed to completely soak through the prefilter. 4. After about 3 min the mixture is completely soaked through the filters and the prefilter is removed and discarded. 5. Results are read directly on the filter and interpreted as follows: • inconclusive: only a round spot (.) is visible; test has to be repeated; • negative: only a dot bar ( . . . ) is visible; and • positive: both a round spot (.) and a dot bar ( . . . ) are visible.
94
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
Table 1 Number of positive and negative results obtained with the rapid test in EIA positive whole and diluted blood samples HCV-positive EIA test
Positive Negative Sensitivity (%)
Whole
Diluted blood
blood
1:20
1:50
1:100
50 0
48 2
15 35
2 48
100
96
30
4
3. Results Results observed in whole and diluted blood are showed in Tables 1 and 2 and refer to the accuracy of the test with respect to the EIA test which was the reference method. Results give evidence of a good sensitivity (expressed by the percentage of positive results in HCV EIA-positive samples) of the rapid test when compared with EIA test up to a 1:20 dilution of the whole blood sample. The specificity (expressed by the percentage of negative results in HCV EIA-negative samples) of the rapid test also gave better results with respect to the sensitivity. Only one false-positive was found in all samples tested.
4. Discussion The diagnosis of HCV infections is most frequently accomplished using EIA methods. The methods are highly accurate but they require sophisticated laboratory equipment and over 2 h for completion. Alternatives to these methods are today available and they can be applied in some particular instances: where Table 2 Number of positive and negative results obtained with the rapid test in EIA negative whole and diluted blood samples HCV-negative EIA test Whole
Diluted blood
blood
1:20
1:50
1:100
Positive Negative
1 49
0 50
0 50
0 50
Specificity (%)
98
100
100
100
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
95
rapid results are required (emergency transfusions and transplantation services, or at the time of an accidental percutaneous inoculation involving a health care worker), where facilities and capabilities are less than optimal (small blood centers or in case of epidemiological studies) or, as a complementary aid, in physician’s offices and dental surgeries in order to reduce the risk of patient-topatient transmission [5–8]. The results of the present research show that the rapid HCV assay can be easily and effectively performed for this purpose. The test is extremely easy to carry out and interpret and a quality control measure ensures that it is performed properly. The results are produced in less than 3 min and only a mechanical pipette is required. Further, contrary to other rapid tests previously described [2–4], the test can be performed on whole blood, thus avoiding sophisticated procedures in order to separate serum from cells. The high sensitivity expressed by the test up to 1:20 dilution of the blood, ensures optimal results even in the presence of blood contaminants, for example oral fluids, and can allow to formulate a precise diagnosis even from saliva samples, provided that little amounts of blood, during spontaneous or provoked gingival bleeding, are included. Therefore, it can represent a useful method for a rapid identification of HCV-positive subjects just before the onset of a dental treatment allowing the dentist to improve the procedures for cross-infection control. Further, the possibility to perform accurate oral fluid testing for HCV antibody should stimulate epidemiological studies on large population groups where reluctance to consent to venipuncture is an impediment to epidemiological evaluation. In addition to the high sensitivity, the method showed a very high specificity considering that only one false-positive was detected in all samples tested. This property is welcome in order to avoid unjustified alarms in case of HCVnegative subjects.
Acknowledgements This work was supported by Ditta Castellini s.p.a.
References [1] Klein RS, Freeman K, Taylor PE, Stevens CE. Occupational risk for hepatitis C virus infection among New York City dentists. Lancet 1991;338:1539–42. [2] Constantine NT, Callahan JC, Watts DM. Successful use of two rapid HCV assays in a high prevalence Romanian population. J Chin Lab Anal 1994;8:332–4.
96
L. Montebugnoli et al. / Clinica Chimica Acta 288 (1999) 91 – 96
[3] Poovorawan Y, Theamboonlers A, Chumdermpadetsuk S, Thong CP. Comparative results in detection of HCV antibodies by using a rapid HCV test, ELISA and immunoblot. Southeast Asian J Trop Med Public Health 1994;25(4):647–9. [4] Mvere D, Constantine NT, Katsawde E, Tobaiwa O, Dambire S, Corcoran P. Rapid and simple hepatitis assay:encouraging results from a blood donor population in Zimbabwe. Bull World Health Organ 1996;74(1):19–24. [5] Chen M, Yun ZB, Sallberg M, Schvarcz R, Bergquist I, Berglund HB. Detection of hepatitis C virus RNA in the cell fraction of saliva before and after oral surgery. J Med Virol 1995;45(2):223–6. [6] Piazza M, Borgia C, Picciotto L, Nappa S, Cicciarello S, Orlando R. Detection of hepatitis C virus-RNA by polymerase chain reaction in dental surgeries. J Med Virol 1995;45(1):40–2. [7] Allander T, Gruber A, Naghavi M. Frequent patient-to-patient transmission of hepatitis C in haematology ward. Lancet 1995;345:603–7. [8] Porter SR, Lodi G. Hepatitis C virus (HCV) — an occupational risk to dentists? Br Dent J 1996;180(12):473–4.