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Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations Leticia de Paula Scalioni a , Helena Medina Cruz a , Vanessa Salete de Paula b , Juliana Custódio Miguel a , Vanessa Alves Marques a , Cristiane Alves Villela-Nogueira c , Flavio Augusto Pádua Milagres d , Marcelo Santos Cruz e , Francisco Inácio Bastos f , Tarcisio Matos Andrade g , Ana Rita Coimbra Motta-Castro h , Lia Laura Lewis-Ximenez a , Elisabeth Lampe a , Livia Melo Villar a,∗ a

Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil Laboratory of Tecnhological Development of Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil Hepatology Division, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil d Medicine Faculty, Federal University of Tocantins, Palmas, Brazil e Institute of Psychiatry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil f Institute of Communication and Scientific Information & Technology for Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil g Department of Community & Family Health, Federal University of Bahia, Salvador, Brazil h Department of Biochemistry and Pharmacy, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil b c

a r t i c l e

i n f o

Article history: Received 24 December 2013 Received in revised form 18 March 2014 Accepted 2 April 2014 Keywords: Hepatitis C virus Diagnosis Rapid test Endemicity Prevalence

a b s t r a c t Background: Rapid tests for the detection of antibodies to hepatitis C virus (anti-HCV) can facilitate access to diagnosis. Objectives: This study aimed to evaluate the performance of rapid tests for anti-HCV detection in the sera, whole blood, and oral fluid samples from individuals with different endemicity profiles and risk behaviors. Study design: Three groups donated biological samples that were tested using three anti-HCV rapid tests (WAMA, Bioeasy and OraSure): (I) suspected cases of hepatitis C, (II) individuals who were living in remote areas in Brazil and (III) crack users and beauty professionals. Reproducibility, repeatability and cross-reactivity to other infectious agents (dengue, HIV, malaria, and syphilis) were also evaluated. Results: In group I, specificities varied from 93.75% to 100% and sensitivities varied from 76.03% to 93.84% according to the EIA results. When anti-HCV/HCV RNA-reactive sera samples were considered truepositive HCV cases, the sensitivities and specificities varied from 86.3% to 99.09% and 93.75% to 100%, respectively. In group II, the OraSure rapid test presented the best performance. In group III, the Bioeasy assay performed best using saliva and whole blood and the OraSure assay performed best using oral fluid samples. The reproducibility and repeatability of the WAMA and Bioeasy tests were excellent. The level of concordance between the HCV EIAs and the rapid tests using samples that were reactive for other infectious agents varied from 82.35% to 100% for the WAMA assay and 94.11% to 100% for the Bioeasy assay. Conclusion: All of the rapid tests could be used to identify active HCV infection among individuals with different endemicity profiles and risk behaviors. © 2014 Elsevier B.V. All rights reserved.

1. Background

∗ Corresponding author at: Viral Hepatitis Laboratory, Helio and Peggy Pereira Pavillion, Ground Floor, Room B09, FIOCRUZ Av. Brasil, 4365, Manguinhos, 210360040 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1918. E-mail addresses: [email protected]fiocruz.br, liviafi[email protected] (L.M. Villar).

The hepatitis C virus (HCV) infects approximately 130–170 million people worldwide [1]; however, more than half of HCVinfected persons are unaware of their status [2]. Since 1998, the CDC has recommended that persons whose are at risk of infection routinely undergo anti-HCV testing [3].

http://dx.doi.org/10.1016/j.jcv.2014.04.001 1386-6532/© 2014 Elsevier B.V. All rights reserved.

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A diagnosis of HCV infection is based on the detection of antigens, antibodies or the viral genome in serum or plasma samples. An enzyme immunoassay (EIA) is highly sensitive for the detection of antibodies to HCV (anti-HCV) in at-risk individuals [4–7]. Rapid tests may be suitable for resource-limited settings because they are cost-effective and simple to perform, and these tests provide same-day results. HCV rapid tests have been evaluated in highrisk individuals who reside in large urban centers, such as injecting drug users, and the sensitivities have varied from 90.8% to 99.9% and the specificities have varied from 92.1% to 99.9% [8–11]. In Brazil, these assays were recently evaluated; however, few samples were included in the studies and all of the samples were HCV RNA-reactive [12]. A rapid HCV assay could increase testing opportunities outside of traditional laboratory settings, such as in clinics and physician offices. However, the performance of commercially available rapid HCV antibody tests should be evaluated according to different biological specimens (serum, whole blood and oral fluid) and settings (high- and low-risk populations). 2. Objectives The aim of this study was to determine the performance of antiHCV rapid assays using biological samples from individuals who resided in areas with a low or high HCV prevalence. 3. Study design 3.1. Biological samples 3.1.1. Reference panel A panel of 575 serum samples was obtained from individuals who were referred to the Viral Hepatitis Laboratory (IOC/FIOCRUZ, Rio de Janeiro) and the Hospital Clementino Fraga Filho (UFRJ, Rio de Janeiro). In this group, EIAs from two different manufacturers [(ETIAB-HCVK-4, Diasorin, Vercelli, Italy and HCV Ab, Radim, Pomezia, Italy)] were used because they include different capture antigens. The Radim EIA detects the binding of HCV antibodies in the sample to specific antigens (core and recombinant peptides, such as NS3, NS4 and NS5) that comprise the solid phase of the plate. In the Diasorin EIA, HCV antigens from structural and non-structural regions were presented in the solid phase; however, the specific HCV antigens were not available in the manufacturer’s instructions. Thereafter, all anti-HCV-reactive samples as detected by both EIAs were retested using an EIA in duplicate and submitted to the COBAS AMPLICOR HCV Test 2.0 (Roche Diagnostics, Pleasanton, CA, USA) to confirm the presence of HCV RNA. This algorithm (initial testing for HCV antibodies, followed by HCV RNA testing) is recommended by the CDC to diagnose HCV infections and to confirm EIA data [13]. Additionally, HCV RNA-reactive samples were submitted to HCV genotyping using the Versant HCV Genotype 2.0 Assay (Siemens, New York, USA). 3.1.2. Field-based studies From February 2010 to September 2011, biological samples were obtained from volunteers who represented the following 3 populations: (group I) a population with a high anti-HCV prevalence, (group II) a population with a low anti-HCV prevalence, and (group III) a population with a high risk of becoming infected with HCV. Group I was composed of individuals who were referred to Viral Hepatitis Ambulatory Clinics in Rio de Janeiro, which were considered settings with a high HCV prevalence. According to the Brazilian Health Ministry, approximately 70,000 cases of chronic

hepatitis C occurred from 1999 to 2010 and more than half (47,830 individuals) of the cases were from the Southeast region of the country (where Rio de Janeiro is located) [14]. In 2010, the average number of cases was 4.5 cases per 100,000 inhabitants in Brazil and the Southeast region had 6.8 cases per 100,000 inhabitants [14]. Group II was composed of individuals who were living in remote areas in the North and Mid-west Regions of Brazil (Tocantins and Mato Grosso do Sul States), which were considered settings with a low prevalence of HCV. According to the Brazilian Health Ministry, the anti-HCV prevalence rates were 3.2 cases per 100,000 inhabitants in Mato Grosso do Sul and 0.5 cases per 100,000 inhabitants in Tocantins State [14]. Group III was composed of individuals with a high risk of being exposed to HCV (crack users in the Southeast and Northeast regions of Brazil and beauty professionals who were living in the Rio de Janeiro State). In Brazil, the anti-HCV prevalence among crack users has ranged from 1.3% to 27.7% [15–17] and this prevalence among beauty professionals was 2% [18]. In group I, the inclusion criteria included individuals who were more than 18 years of age and who read and signed the informed consent form, individuals who were either symptomatic or asymptomatic for hepatitis but had one or more risk factors for HCV infection (a history of intravenous drug use, sexual intercourse with a known carrier of hepatitis C, sexually transmitted disease, longterm hemodialysis, surgery or blood transfusion before 1994, or piercing). In group II, the inclusion criteria included individuals who read and signed the informed consent form and individuals whose parents or legal guardians read and signed the informed consent form. In group III, the inclusion criteria included crack users who used drugs within the previous 12 months and beauty professionals, including manicurists, pedicurists, and hairdressers, who worked during the previous 30 days before sample collection. This study was approved by the Ethics Committee of FIOCRUZ (636/09). Informed consent was obtained from each patient who was included in the study, and the study protocol was followed according to the ethical guidelines of the 1975 Declaration of Helsinki. Blood samples were collected by venipuncture to obtain whole blood and sera samples. Oral fluid samples were collected using the commercial device Salivette (Sarstedt, Rommelsdorf, Germany) and OraQuick swabs (OraSure Technologies, Inc., Bethlehem, Pennsylvania) as previously described [8,9,19,20].

3.2. Anti-HCV rapid tests The following 3 rapid tests were evaluated in this study: (1) the WAMA Imuno-Rápido HCV Kit (WAMA Diagnóstica, São Carlos, São Paulo, Brazil), (2) the Bioeasy HCV Rapid Test (Bioeasy Diagnóstica Ltda, Belo Horizonte, Minas Gerais, Brazil) and (3) the OraQuick HCV Rapid Test (OraSure Technologies, Bethlehem, Pennsylvania, USA). All of the tests were single-use, disposable-chamber, in vitro, qualitative, immune-chromatographic assays that could detect anti-HCV antibodies and provide visual results in less than 40 min. The principle of these assays was similar. Test strips contained recombinant antigens from the core and non-structural regions of the HCV genome, which was immobilized. The rapid tests were conducted according to the manufacturer’s instructions: 10 ␮l of serum or whole blood was used for the WAMA and Bioeasy assays and 20 ␮l of oral fluid was used in these assays. Reactive results may be observed after 10–15 min using the WAMA assay, 5–20 min using the Bioeasy test, and 40 min using the OraSure rapid test. For the WAMA and Bioeasy assays, the results should not be considered after 20 min.

Please cite this article in press as: Scalioni LP, et al. Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.001

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3.3. Serological and molecular assays

4. Results

The sera samples from the reference panel were tested as previously described. In groups I, II, and III of the field study, the Radim EIA was used to test the sera samples because the manufacturer described the HCV antigens that were used for capture. Reactive anti-HCV sera samples were tested using the COBAS AMPLICOR HCV Test 2.0 (Roche Diagnostics, Pleasanton, CA, USA) to confirm the data when the sample volume was available [13]. HCV RNAreactive samples were genotyped using the Versant HCV Genotype Assay 2.0 (Siemens).

4.1. Evaluation of the HCV rapid tests using reference panels

3.4. Performance of the OraQuick HCV antibody rapid test in serum, whole blood and oral fluid samples To evaluate the OraQuick HCV Rapid Antibody Test in laboratory conditions using whole blood, serum and oral fluid samples, a convenience sample of 120 individuals was recruited from the Centers for Viral Hepatitis in Rio de Janeiro. In this convenience sample, approximately 60% of the individuals presented with antiHCV/HCV RNA-reactive serum and 40% of individuals did not have an HCV marker. The inclusion criteria for these patients were the same as those for group I in the field study. Therefore, 81 individuals with samples that were anti-HCV/HCV RNA-reactive and 39 individuals with samples that were not anti-HCV-reactive were included. The mean age was 50.52 (±13.40) years, and 54.1% (65/120) of the participants were female. Among the HCV cases, 9 individuals had been treated, 6 were on treatment, 37 had not been treated and 29 did not provide any information. Biological samples were obtained as previously described. 3.5. Reproducibility and repeatability evaluation To evaluate the reproducibility and repeatability of the anti-HCV Bioeasy and WAMA rapid tests, 4 samples (2 serum and 2 saliva samples) were tested by two different operators for 2 consecutive days, of which 1 serum sample and 1 saliva sample were antiHCV-reactive and 1 serum sample and 1 saliva sample were not anti-HCV-reactive. The agreement was calculated by comparing the rapid test results with the EIA results. 3.6. Cross-reactivity Cross-reactivity for other viral, bacterial and protozoan infections was evaluated. Sera samples that were reactive for dengue virus (DENV) (n = 35), human immunodeficiency virus (HIV) (n = 30), Plasmodium vivax (n = 17) and Treponema pallidum (n = 20) were kindly donated by reference centers located in Rio de Janeiro, Brazil. These samples were tested for anti-HCV using ELISA (HCV Ab, Radim, Italy) and the Bioeasy and WAMA rapid tests. 3.7. Data analysis Clinical and epidemiological data were coded and entered into a database (Excel 2010, Microsoft Inc., USA), and SPSS for Windows, version 20.0 (SPSS Inc., USA) was used for the statistical analysis. The detection of anti-HCV antibodies in the serum samples by the EIAs was used as the gold standard for the assessment of the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV) of each rapid test. To evaluate the deviation between the observed and expected values in the groups, the 2 test or Fisher’s exact test were used. The Kappa coefficient (k) was used to assess the degree of agreement between the reference panel and the rapid tests. Two-tailed P-values < 0.05 were considered statistically significant.

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The reference panel was composed of 575 individuals, of which 251 exhibited anti-HCV antibodies in serum and 324 did not exhibit anti-HCV antibodies in serum according to the EIAs. In addition, 240 individuals had anti-HCV reactivity according to the Bioeasy assay and 234 individuals had anti-HCV reactivity according to the WAMA assay, which were associated with sensitivities of 95.62% and 93.23%, respectively. Among 251 sera samples that were anti-HCV-reactive according to the EIAs, 202 had HCV RNA, which mostly belonged to genotype 1 (53.5%). When the antiHCV/HCV RNA-reactive samples were considered, the sensitivities were 98.51% for the Bioeasy assay and 97.03% for the WAMA assay (Table 1). The sera samples that were false-negative for anti-HCV antibodies according to the Bioeasy and WAMA assays had a low optical density to cut-off value ratio (OD/CO) in the EIAs when compared with samples that were true-positive for anti-HCV antibodies according to both assays (data not shown). 4.2. Evaluation of the HCV rapid tests using a field study A field-based study was conducted, which included 824 volunteers with a mean age (±SD) of 34.89 (±17.80) years and 51.6% of the participants were female. These volunteers were divided into the following 3 groups: (group I) 194 suspected cases of hepatitis C, (group II) 430 individuals from low HCV prevalence settings that were located in remote areas in the North and Mid-West Regions of Brazil (Tocantins and Mato Grosso do Sul States), and (group III) 200 individuals with a high risk of being exposed to HCV (crack users from the Southeast and Northeast regions of Brazil and beauty professionals who were living in the Rio de Janeiro State). To evaluate the OraQuick HCV Rapid Test, a subgroup of 674 individuals with a mean age of 33.08 (±18.13) years and a predominance of women (347/674) were included. These individuals were divided into residents of remote areas (Tocantis State) (n = 459), crack users (n = 43) and suspected HCV cases (n = 172). In group I, the overall specificities varied from 93.75% to 100% and the overall sensitivities varied from 76.03% to 93.84% using the Bioeasy and WAMA assays and considering only the EIA results (Table 2). When the anti-HCV/HCV RNA-reactive sera samples were considered true-positive HCV cases, the sensitivities and the specificities increased for both assays. The highest concordance was observed for the Bioeasy assay when serum and whole blood samples were used; however, this test performed worst using saliva samples. In groups II and III, anti-HCV-reactive samples were not found using the rapid tests and it was not possible to determine the sensitivities of these assays. The OraQuick HCV Rapid Test performed best in both groups, whereas the Bioeasy assay performed best using the saliva and whole blood samples from crack users and beauty professionals (Table 3). In low HCV prevalence settings, 7 samples were anti-HCVreactive according to ELISA and 4 of them were submitted to RT-PCR; however, HCV RNA was not detected. Three anti-HCVreactive samples were not tested using the COBAS AMPLICOR HCV Test 2.0 due to a low sample volume. 4.3. Performance of the OraQuick HCV antibody rapid test in serum, whole blood and oral fluid samples The OraQuick HCV antibody rapid test performed well in laboratory settings using all of the biological specimens. The highest accuracy (100%) was achieved using sera samples, followed by

Please cite this article in press as: Scalioni LP, et al. Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.001

(b)

TN

FP

FN

n

Sensitivity (95% CI)

240 234

324 321

0 3

11 17

575 575

95.62% (92.29%–97.79%) 93.23% (89.38%–96.01%)

Anti-HCV- and HCV RNA-reactive Bioeasy HCV Rapid Test 199 WAMA Imuno-Rápido HCV Kit 196

324 321

0 3

3 6

526 526

98.51% (95.72%–99.69%) 97.03% (93.65%–98.90%)

Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

K

100% (98.87%–100%) 99.07% (97.32%–99.81%)

100% (98.47%–100%) 98.73% (96.35%–99.74%)

96.73% (94.22%–98.35%) 94.97% (92.07%–97.04%)

96.09% (93.8%–98.3%) 92.88% (89.82%–95.94%)

100% 98.87%–100%) 99.07% (97.32%–99.81%)

100% (98.16%–100%) 98.49% (95.66%–99.69%)

99.09% (97.35%–99.81%) 98.17% (96.05%–99.32%)

98.79% (97.43%–100%) 96.37% (94.02%–98.72%)

P < 0.0001; Note. TP, true-positive; TN, true-negative; FP, false-positive; FN, false-negative; n, number of samples; CI, confidential interval; PPV, positive predictive value; NPV, negative predictive value; K, kappa.

Table 2 Performance of HCV rapid tests using saliva, whole blood and serum samples from individuals in high HCV prevalence settings. (a) Performance of HCV rapid tests using all of the samples that were anti-HCV-reactive. (b) Performance of HCV rapid tests using only those samples that were anti-HCV/HCV RNA-reactive. TP

TN

FP FN

N

Sensitivity (95% CI)

Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

K

(a) Anti-HCV-reactive Bioeasy HCV Rapid Test/serum samples WAMA Imuno-Rápido HCV Kit/serum samples Bioeasy HCV Rapid Test/whole blood samples Bioeasy HCV Rapid Test/saliva samples WAMA Imuno-Rápido HCV Kit/saliva samples OraQuick HCV Rapid Test/saliva samples

Rapid test/sample

136 134 137 111 119 108

48 45 48 48 45 50

0 3 0 0 3 0

10 12 9 35 45 14

194 194 194 194 194 172

93.15% (87.76%–96.67%) 91.78% (86.08%–95.68%) 93.84% (88.62%–97.14%) 76.03% (68.27%–82.70%) 81.51% (74.25%–87.44%) 88.52% (81.50%–93.58%)

100% (92.60%–100%) 93.75% (82.80%–98.69%) 100% (92.60%–100%) 100% (92.60%–100%) 93.75% (82.80%–98.69%) 100% (92.89%–100%)

100% (97.32%–100%) 97.81% 93.73%–99.55%) 100% (97.34%–100%) 100% (96.73%–100%) 97.54% (92.98%–99.49%) 100% (96.64%–100%)

82.76% (70.57%–91.41%) 78.95% (66.11%–88.62%) 84.21% (72.13%–92.52%) 57.83% (46.49%–68.60%) 62.5% (50.30%–73.64%) 78.13% (66.03%–87.49%)

87.06% (79.25%–94.87%) 80.47% (70.98%–89.96%) 88.28% (80.8%–95.76%) 61.08% 49.41%–72.75%) 64.44% (52.74%–76.14%) 81.77% (72.62%–90.92%)

(b) Anti-HCV and HCV RNA-reactive Bioeasy HCV Rapid Test/serum samples WAMA Imuno-Rápido HCV Kit/serum samples Bioeasy HCV Rapid Test/whole blood samples Bioeasy HCV Rapid Test/saliva samples WAMA Imuno-Rápido HCV Kit/saliva samples OraQuick HCV Rapid Test/saliva samples

109 108 109 95 100 82

48 45 48 48 45 50

0 3 0 0 3 0

1 2 1 15 10 4

158 158 158 158 158 136

99.09% (95.04%–99.98%) 98.18% (93.59%–99.78%) 99.09% (95.04%–99.98%) 86.36% (78.51%–92.16%) 90.91% (83.92%–95.55%) 95.35% (88.52%–98.72%)

100% (92.60%–100%) 93.75% (82.80%–98.69%) 100% (92.60%–100%) 100% (92.60%–100%) 93.75% (82.80%–98.69%) 100% (92.89%–100%)

100% (96.67%–100%) 97.30% (92.30%–99.44%) 100% (96.67%–100%) 100% (96.19%–100%) 97.09% (91.72%–99.40%) 100% (95.60%–100%)

97.96% (89.15%–99.95%) 95.74% (85.46%–99.48%) 97.96% (89.15%–99.95%) 76.19% (63.79%–86.02%) 81.82% (69.09%–90.92%) 92.59% (82.11%–97.94%)

98.51% (95.6%–100%) 92.47% (85.98%–98.96%) 98.51% (95.6%–100%) 79.37% (69.44%–89.3%) 81.32% (71.59%–91.05%) 93.78% (87.77%–99.79%)

p < 0.0001; Note. TP, true-positive; TN, true-negative; FP, false-positive; FN, false-negative; n, number of samples; CI, confidential interval; PPV, positive predictive value; NPV, negative predictive value; K, kappa.

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Table 1 Performance of rapid assays for the detection of anti-HCV antibodies in serum samples using two commercial immunoenzymatic assays (ETI-AB-HCVK-4, Diasorin, and HCV Ab, Radim). (a) Performance of HCV rapid tests using all of the samples that were anti-HCV-reactive (n = 575). (b) Performance of HCV rapid tests using only those samples that were anti-HCV-reactive and had HCV RNA (n = 526).

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Table 3 Performance of HCV rapid tests according to specimen type in two different populations: individuals with risk behavior and individuals from low HCV prevalence settings. Rapid test/sample

N

Population with risk behavior TP

Bioeasy HCV Rapid Test/serum WAMA Imuno-Rápido HCV Kit/serum Bioeasy HCV Rapid Test/whole blood Bioeasy HCV Rapid Test/saliva WAMA Imuno-Rápido HCV Kit/saliva OraQuick HCV Rapid Test/saliva

200 200 200 200 200 43

0 0 0 0 0 0

TN 199 198 200 200 198 43

n

FP

FN

1 2 0 0 2 0

0 0 0 0 0 0

430 430 430 430 430 459

Population from low HCV prevalence settings TP

TN

FP

FN

0 0 0 0 0 0

421 423 423 422 423 455

2 0 0 1 0 0

7 7 7 7 7 4

Note. TP, true-positive; TN, true-negative; FP, false-positive; FN, false-negative; n, number of samples; CI, confidential interval; PPV, positive predictive value; NPV, negative predictive value; K, kappa.

whole blood samples (98.77%) and oral fluid samples (97.53%). No false-positive results were observed using the OraSure assay in this evaluation (Table 4). 4.4. Reproducibility and repeatability evaluation A total of 176 tests were performed, of which 88 used serum and 88 used saliva samples. In this evaluation, 5 Bioeasy rapid tests presented invalid results using saliva samples. The WAMA and Bioeasy assays exhibited 100% concordance with the EIA results for all of the samples (sera and saliva samples were HCV-reactive or non-reactive). 4.5. Cross-reactivity to other viral, protozoan and bacterial infections Samples that were reactive for HIV, dengue virus, P. vivax and T. pallidum were evaluated using the anti-HCV rapid tests and EIAs. Using the EIAs, anti-HCV-reactive samples were detected among samples that were positive for HIV (n = 1/30), DENV (n = 1/35), P. vivax (n = 1/17), and T. pallidum (n = 8/20). The level of concordance between the HCV EIAs and the rapid tests varied from 82.35% to 100% using the WAMA assay and 94.11% to 100% using the Bioeasy assay. False-negative results were obtained in 3 samples using the Bioeasy HCV Rapid Test [DENV (n = 1), HIV (n = 1) and P. vivax (n = 1)] and in 2 samples using the WAMA Imuno-Rápido HCV Kit [DENV (n = 1) and HIV (n = 1)]. Moreover, 3 false-positive anti-HCV results were obtained among P. vivax-reactive samples using the WAMA Imuno-Rápido HCV Kit. No discordant results were obtained among the EIAs and the rapid tests using T. pallidum-reactive samples. 5. Discussion This study demonstrated the utility of the HCV rapid tests for the detection of anti-HCV antibodies in laboratory and field settings. Using the reference serum panel, a high concordance was observed between the rapid test and EIA results; however, the samples with a low OD/CO ratio as detected by the EIAs tended to present falsenegative results in the rapid tests. In addition, the sensitivities and the specificities increased when only the anti-HCV/HCV RNAreactive samples were included as previously observed [12].

In the high HCV prevalence group, the Bioeasy assay performed the best, most likely due to the presence of recombinant antigens from core and non-structural regions of the HCV genome, which were immobilized on test strips. Using whole blood, the Bioeasy assay had a sensitivity that was similar to that of the Chembio DDP HCV Test (Chembio Diagnostic Systems, Inc, USA) (96.2%) and that of the OraQuick Rapid HCV Antibody Test (95.9%) [11]. Moreover, the good efficiency of the Bioeasy assay due to the anti-HCV/HCV RNA-reactive samples suggests that rapid tests can efficiently detect active HCV infection, which may improve access to early diagnosis and treatment for these patients. False-negative anti-HCV serum samples as determined by rapid tests usually do not exhibit HCV RNA, which was observed in the evaluation of the OraQuick HCV (OraSure) Rapid Test using oral fluid and whole blood samples [7,10]. In the individuals from low HCV prevalence settings and individuals who presented a risk behavior, the OraQuick Rapid HCV Antibody Test performed the best using oral fluid samples. Additionally, the Bioeasy HCV Rapid Test performed well using each type of biological specimen from individuals who presented a risk behavior. However, false-negative results were observed in this group, which has been previously observed for the Chembio, MedMira and OraSure rapid tests using samples from intravenous drug users [11,21] possibly due to low analytic concentrations. In this study, oral fluid samples were evaluated as an alternative fluid for the detection of anti-HCV antibodies using rapid tests. The OraQuick HCV Rapid Antibody Test was associated with the best performance using these samples, which is a similar finding to those in other studies [8,9]. This assay was evaluated under laboratory conditions, and the best performance was achieved using sera samples, which was previously observed by Cha et al. [22]. This finding may be explained by the low concentration of antiHCV antibodies that is present in oral fluid compared with blood or serum, which has been observed in anti-HCV rapid tests and EIAs using oral fluid samples [8,19,21,22]. The WAMA and Bioeasy assays had excellent reproducibility and repeatability, which demonstrates the utility of these assays for an HCV diagnosis. In the cross-reactivity analysis, false-negative anti-HCV results were observed using the rapid tests to evaluate anti-HIV and dengue-reactive samples, which may be due to the decreased production of anti-HCV antibodies during HIV infection [21,23] or dengue virus infection or cross-reactivity in the EIAs. Among the P. vivax-reactive samples, false-negative and

Table 4 Performance of the OraQuick HCV Rapid Test using 120 paired serum, whole blood and oral fluid samples that were evaluated under laboratory conditions. Sample

TP

TN

FP

FN

Serum Whole blood Oral fluid

81 80 79

39 39 39

0 0 0

0 1 2

Sensitivity (95% CI) 100% (95.55%–100%) 98.77% (93.31%–99.97%) 97.53% (91.36%–99.70%)

Specificity (95% CI)

PPV (95% CI)

PNV (95% CI)

K (%)

100% (90.97%–100%) 100% (90.97%–100%) 100% (90.97%–100%)

100% (95.55%–100%) 100% (95.49%–100%) 100% (95.44%–100%)

100% (90.97%–100%) 97.5% (86.84%–99.94%) 95.12% (83.47%–99.40%)

100 98.11 96.25

Note. TP, true-positive; TN, true-negative; FP, false-positive; FN, false-negative; n, number of samples; CI, confidential interval; PPV, positive predictive value; NPV, negative predictive value; K, kappa.

Please cite this article in press as: Scalioni LP, et al. Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.001

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false-positive results were observed. In addition, false-positive anti-HCV EIA and RIBA results were observed among samples that were reactive for P. falciparum [24]. No discordant results were observed using T. pallidum-reactive samples. This study has several limitations, such as the insufficient sample volume to conduct HCV RNA testing in several anti-HCVreactive samples in the field study. In addition, the OraQuick HCV test could not be used to analyze serum and whole blood in the field study or in the reproducibility and cross-reactivity analyses due to the low sample volume and the high costs of this test in the Brazilian market because this product must be imported. In conclusion, all of the rapid tests could be used to identify active HCV infection and may be a useful tool to increase testing opportunities outside of traditional laboratory settings, such as in clinics, community outreach centers, physician offices and especially remote areas in developing countries, including Brazil. Funding This research was supported by the Fundac¸ão de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Brazilian National Counsel of Technological and Scientific Development (CNPq), the Coordination of Improvement of Higher Education Personnel (CAPES) and the Oswaldo Cruz Foundation (FIOCRUZ). Competing interests The authors disclose no actual or potential conflicts of interest, including any financial, personal or other relationships with people or organizations within two years of the beginning of this study that could inappropriately influence the study. Ethical approval This study was approved by the Ethics Committee of FIOCRUZ (636/09). Informed consent was obtained from each patient who was included in the study, and the study protocol was followed according to the ethical guidelines of the 1975 Declaration of Helsink. Acknowledgments The authors would like to thank Jaqueline Correia de Oliveira, Brunna Lemos Crespo Marques, Elisangela Ferreira da Silva, Moyra Machado Portilho, Renata Tourinho Santos, and Paula Guerra Murat for technical assistance; Dr. José Henrique Pilotto, Dr. Martha Murtez and Dr. Flavia Barreto dos Santos who provided samples that were reactive for HIV, P. vivax and dengue virus. References [1] Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011;17:107–15. [2] Southern WN, Drainomi ML, Smith BD, Christiansen CL, McKee D, Gifford AL, et al. Hepatitis C testing practices and prevalence in high-risk urban ambulatory care setting. J Viral Hepat 2011;18:474–81.

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Please cite this article in press as: Scalioni LP, et al. Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.001

Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations.

Rapid tests for the detection of antibodies to hepatitis C virus (anti-HCV) can facilitate access to diagnosis...
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