Dried blood spot for hepatitis C virus serology and molecular testing

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

We investigated the performance of dried blood spots (DBS) in hepatitis C virus (HCV) diagnosis using modified commercial tests. Paired DBS and serum samples were collected from 200 patients: 100 patients with anti-HCV antibodies (anti-HCV), including 62 patients with detectable serum HCV RNA, and 100 patients without anti-HCV. The DBS sample consisted of three drops of approximately 50 μL of whole blood applied to a paper card, which was then stored at −20°C within 48 hours of collection. Using the Ortho HCV 3.0 enzyme-linked immunosorbent assay kit on DBS, we observed both a specificity and sensitivity of 99% in detecting anti-HCV. HCV RNA was detected on DBS in 60/62 (97%) patients with detectable serum HCV RNA, which was then successfully quantified in 55 samples (89%) using the Cobas TaqMan HCV test. A good correlation was observed between the DBS HCV RNA concentration and the serum level (r2 = 0.95; P < 0.001). HCV genotyping was successfully performed on DBS samples, with a full concordance between the 14 paired DBS and serum samples (genotypes 1-4). Conclusion: This study presents DBS as a reliable alternative to serum specimens for detecting anti-HCV, quantifying HCV RNA and genotyping HCV. DBS may increase the opportunities for HCV testing and treatment follow-up in hard-to-reach individuals. (HEPATOLOGY 2010.)

Hepatitis C virus (HCV) infection is currently underdiagnosed, leaving many individuals unaware of their infection status. Some population groups, such as sex workers, the homeless, prisoners, or other institutionalized individuals, have a higher prevalence of HCV infection than the general population.1–3 However, HCV testing in these groups is limited by the poor acceptability or feasibility of venipuncture. Collecting capillary blood spots on filter paper requires less staff training, is less invasive, involves smaller blood volumes, and is ideal for high-risk patients with damaged veins, such as intravenous drug users.4 In addition, this technique can reduce the cost of HCV testing by simplifying sample collection, processing (no centrifugation), storage, and shipment. Many studies have already demonstrated the value of dried blood spots (DBS) for the serological and molecular diagnosis of human immunodeficiency virus.5–11 Routine screening for HCV infection relies on detecting antibodies against HCV (anti-HCV) using highly sensitive second- or third-generation enzyme immunoassay. DBS have been used to detect anti-HCV among intravenous drug users, prisoners and childbearing women.12–17 However, the diagnosis of acute or chronic infection also requires the detection of HCV RNA by polymerase chain reaction (PCR).18, 19 Likewise, when recent HCV infection is suspected or the patient is immunocompromised, then the sample should be referred for PCR. Samples with a low screening signal-to-cutoff ratio may also need confirmation with these more specific recombinant immunoblot assays.20, 21

In this study, we have investigated both anti-HCV and HCV RNA detection and quantification from DBS samples collected under different storage conditions using commercially available in vitro diagnostic assays and fully automated viral nucleic acid extraction. Finally, this study also examined the feasibility of HCV genotyping from DBS samples.

Abbreviations

anti-HCV, anti–hepatitis C virus antibody; DBS, dried blood spot; ELISA, enzyme-linked immunosorbent assay; HCV, hepatitis C virus; PCR, polymerase chain reaction.

Materials and Methods

Anti-HCV–Positive and –Negative Specimens.

Samples were collected from patients already attending Montpellier University Hospital for HCV infection diagnosis or monitoring. The study included 100 anti-HCV–positive serum samples and 100 anti-HCV–negative serum samples, with paired DBS. The presence of anti-HCV was confirmed by at least two different enzyme immunoassay techniques on two separate samples. Among them, 62 samples from chronically HCV-infected patients were selected on the basis of detectable serum HCV RNA using the Cobas AmpliPrep/Cobas TaqMan HCV test (Roche Molecular Systems, Branchburg, NJ). The readers of results obtained from DBS were blinded to those from serum.

DBS Preparation.

Fifty microliters of whole blood was absorbed onto a Protein Saver 903 Card (Whatman, Dassel, Germany) to completely fill 12-mm preprinted circular paper disks. These DBS cards were then dried for 18 hours at room temperature, placed in individual zipped plastic bags with a Drierite Desiccant (Fisher Scientific, Illkirch, France) and stored at −20°C for 1 to 8 weeks until use. The stability of anti-HCV and HCV RNA on DBS was also investigated by varying the room temperature exposure from 2 to 12 days before storage at −20°C, and then processing samples in duplicate for HCV RNA quantification and anti-HCV detection.

DBS Anti-HCV Elution and Testing.

Anti-HCV titers were tested using the Ortho HCV 3.0 enzyme-linked immunosorbent assay (ELISA) Test System with Enhanced SAVe kit (Ortho Clinical Diagnostics, Raritan, NJ). A 6-mm disc was punched from the DBS on filter paper. The disc was transferred into an uncoated microtiter plate well for overnight incubation with 200 μL sample diluent with gentle shaking. The next day, the DBS eluate was tested for anti-HCV with a slight modification: instead of 20 μL serum plus 200 μL sample diluent, we used 100 μL of DBS eluate. All subsequent steps, including the interpretation guidelines, were strictly observed. The immunoblot assay INNO-LIA HCV Score (Innogenetics, Ghent, Belgium) was used as a confirmatory test for the presence of anti-HCV. We used 100 μL of the same eluate obtained with sample diluent from the Ortho HCV kit at the first step of the assay, and then followed the manufacturer's recommendations for all subsequent steps.

HCV RNA Extraction and Quantification.

Virus elution and RNA extraction occurred 2 to 8 weeks after DBS storage at −20°C. Elution from DBS was performed using two 6-mm spots cut from the 12-mm preprinted circle by a puncher. The pieces were suspended in a 1.5-mL Eppendorff microtube with 400 μL of buffer prepared extemporaneously (phosphate-buffered saline, 0.05% Tween 20, and 10% bovine serum albumin) and incubated at 4°C for approximately 2 hours under continuous agitation. After centrifugation (20 seconds at 13,000g), the supernatant was collected and RNA was extracted using the automated Cobas Ampliprep Total Nucleic Acid Isolation 100 kit (Roche Diagnostics, Mannheim, Germany). The internal quantification standard (50 μL) was added to each sample before extraction according to the manufacturer's instructions. The internal quantification standard is an internal control of extraction and amplification that also allows HCV quantification.

HCV RNA was quantified using the Cobas TaqMan HCV test (HCV HPS), and the real-time PCR COBAS TaqMan 48 instrument COBAS Ampliprep analyzer (Roche Diagnostics, Mannheim, Germany). Samples were processed in accordance with the manufacturer's instructions, with a 15-IU detection limit. HCV RNA detection below this threshold value was considered as positive but unquantifiable, as recommended by the manufacturer. Specific TaqMan probes were used to detect and assay target and internal quantification standard amplified DNA in each sample. Results were displayed on a computer using the Amplilink 3.01 system connecting the COBAS TaqMan 48 and COBAS Ampliprep instruments.

Genotyping by Sequencing.

Reverse-transcription PCR was performed using the following primers: 5'-GCA GAA AGC GTC TAG CCA TGG CG-3' (sense) and 5'-GAC TCG CAA GCA CCC TAT CAG GCA GT-3' (antisense) and the One Step reverse-transcription PCR kit from Qiagen (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The amplified product was sequenced using the TRUGENE HCV 5' NC kit with the OpenGene DNA sequencing System as recommended by the manufacturer (Siemens Healthcare Diagnostics, Tarrytown, NJ).

Statistical Analysis.

We used the nonparametric Wilcoxon paired-sample test to compare anti-HCV from DBS exposed at room temperature for various time periods. The correlation between DBS and serum HCV RNA levels was calculated with a linear regression analysis after a logarithmic (base 10) transformation to normalize the data. A value equal to 12 IU/mL was arbitrarily allocated to samples positive for HCV RNA but with values below the quantification threshold. To assess the titer result agreement between these two transformed measures, we used the Bland-Altman approach where differences between the two sets of measurements (d i= x i1− x i2) are plotted against mean values (x i1+ x i2)/2. For a Gaussian d i distribution, the limits of agreement are equal to the mean (di) ± 1.96 standard deviation. The slope of the regression line of the differences between the two sets of measurements against the mean values gives the proportional bias, which can be corrected according to the linear regression equation. In the absence of proportional bias or after correction, the mean difference between the two sets of measurements gives the fixed bias. The independence of the residuals was tested after correction.

Statistical analyses were performed using SAS statistical software (version 9, SAS Institute, Cary, NC). Significance was set at P < 0.05.

Results

Sensitivity and Specificity of Anti-HCV Testing on DBS.

Among the 200 DBS samples, the optical density means ± standard deviation were 2.76 ± 1.06, and 0.057 ± 0.053 (P < 0.001) in anti-HCV–positive and –negative groups, respectively (Fig. 1). The threshold value was established using the manufacturer's instructions for serum specimens and corresponded to a mean value of 0.380. This cutoff value was associated with a sensitivity of 99% (95% confidence interval 0.97-1.00) and a specificity of 98% (95% confidence interval 0.97-0.99). One DBS test provided a false negative result with an optical density of 0.112. This specimen was collected from a patient infected with human immunodeficiency virus type 1. In this sample, anti-HCV was detected in serum with a low signal-to-cutoff value at 2.7. HCV RNA was not detected in serum, but the presence of anti-HCV was confirmed using an immunoblot assay. Another patient was anti-HCV–positive on DBS but anti-HCV–negative in serum testing, with a low DBS signal, close to the cutoff value (optical density = 0.404). We considered this as a likely false positive by DBS-based serology.

Figure 1.

Detection of anti-HCV by way of ELISA using dried spotting. Optical density is indicated for patients positive (□) or negative (⋄) for serum anti-HCV antibodies. The dotted lines represent the mean positive cutoff value (0.380).

Serial dilutions of seven DBS and serum samples positive for anti-HCV, diluted in negative blood and serum, respectively, were run in duplicate to investigate the assay's capacity to detect low levels of anti-HCV. The lower limit of anti-HCV detection in DBS was reached at a median of 1/512 titer dilution (range, 1/64 to 1/4,096). The lower limit of detection in serum occurred at a median of 1/1,024 dilution (range, 1/512 to 1/65,336), with a higher titer of two to three serial dilutions in serum compared with the matching DBS.

We compared anti-HCV DBS testing following different storage conditions for eight samples. DBS were exposed at room temperature for 2 to 12 days and tested sequentially. Surprisingly, in HCV-negative samples, the absorbance values reached or exceeded the threshold level with increasing exposure to room temperature (Fig. 2). After 6 days, the absorbance values were greater than the cutoff value in four of the five HCV-negative patient samples. In HCV-positive samples, we did not observe a significant variation in the absorbance value between days 2 and 12 (P = 0.59, Wilcoxon paired test), and all HCV results remained positive.

Figure 2.

Evolution over time of the optical density in anti-HCV antibody testing by way of ELISA. DBS samples were tested after storage at ambient temperature at four time points (48 hours, 6 days, 9 days, 12 days). The dotted line represents the positive cutoff value for anti-HCV antibody detection. Anti-HCV antibody–positive DBS samples are indicated with black symbols; anti-HCV antibody–negative samples are indicated with white symbols.

Anti-HCV Confirmation in DBS Using Recombinant Immunoblots.

Anti-HCV detection controls using ELISA were performed by way of immunoblotting in 10 anti-HCV–positive DBS and 10 anti-HCV–negative DBS and their paired serum samples. Immunoblot results confirmed the results obtained on DBS by way of ELISA. We observed a full concordance between the immunoblot profiles on DBS and serum, with no false-positive or indeterminate results (Fig. 3). We also performed a confirmatory test on the false positive DBS result, which was negative for HCV.

Figure 3.

Confirmation of anti-HCV antibodies using recombinant immunoblotting on DBS and serum specimens. The INNO-LIA HCV Score assay was used to compare the antibody reactivity against HCV proteins on serum and DBS. Results from five representative patients are shown. HCV+, samples positive for anti-HCV antibody detection by way of ELISA. HCV−, sample negative for anti-HCV antibody detection by way of ELISA. S, serum.

Detecting and Quantifying HCV RNA Using DBS.

HCV RNA values obtained from DBS were compared with values from paired serum samples for 62 HCV-infected patients. HCV RNA was detected in 60/62 (97%) of the DBS samples. We measured a low viral load (178 and 331 IU/mL) in the matching serum samples for the two DBS with no HCV RNA detected. Five DBS were positive for HCV RNA but were below the lower limit of detection (15 IU/mL). In matching serum samples, the measured HCV copy number ranged from 334 to 1,672 IU/mL. For 55 samples (89%), DBS successfully quantified HCV RNA, with a range of 477 to 25,000,000 IU/mL in serum (median, 298,000 IU/mL; interquartile range, 25,050 to 1,332,500). Overall, we found a good correlation between DBS and serum HCV RNA measurements (Fig. 4A) (R2 = 0.94; P < 0.0001 [Pearson correlation]).

Figure 4.

Regression analysis of HCV RNA levels and Bland-Altman plot of titer differences in paired DBS and serum samples. (A) Linear regression analysis HCV RNA levels for the 62 matched DBS–serum pairs analyzed by way of real-time PCR quantitative assay. (B) A Bland-Altman analysis was used to determine the agreement between the HCV RNA quantification by plotting the differences between the two sample collections against the averages of the two techniques. The proportional bias observed was corrected in (C) linear regression and (D) Bland-Altman.

We analyzed the differences between the two sample types using a Bland-Altman plot. The mean difference between values (DBS − serum values) was −2.27 log IU/mL ± 0.47, with limits of agreement (mean difference ± 2 standard deviation) of −1.33 and −3.21 log IU/mL (Fig. 4B). Ninety-five percent of the results were within the 95% confidence interval, but we observed a proportional bias. There was a linear relationship between the mean of the two values and their difference, but the slope was significantly different from 0: −0.15 (P < 0.0001). After applying a correction that converts logARN DBS in 1.426 + 1.208 × logARN DBS before calculating the linear regression and the Bland-Altman analysis, the bias disappeared and the concordance became excellent (Fig. 4C,D). To assess the effects of storage temperature, we examined the stability of HCV RNA in DBS stored at room temperature and at −20°C. RNA extraction was performed from DBS after 2, 3, or 6 days. We observed no variation in HCV RNA quantities in samples stored at −20°C. In contrast, samples stored at room temperature demonstrated a dramatic decrease in HCV RNA levels at 6 days (Fig. 5).

Figure 5.

Evolution of HCV RNA value obtained using DBS samples after storage at ambient temperature or −20°C at four time points (6 hours, 48 hours, 3 days, 6 days).

HCV Genotype Characterization in DBS and Serum Samples.

HCV genotypes were determined in 14 randomly selected HCV-infected patients. The HCV RNA viral load in matching serum samples ranged from 2,700 to 1,510,000 IU/mL (median, 337,000 IU/mL). We obtained the genotype sequence in duplicate for all DBS samples. We observed full concordance between DBS and the matching serum samples for the genotypes and subtypes. We found four patients with HCV genotype 1 (1a, n = 1; 1b, n = 2; 1a1b, n = 2), three with genotype 2 (2a/2c, n = 2; unidentified subtype, n = 1), three with genotype 3 (3a, n = 3), and three with genotype 4 (4c, n= 2; 4c/4d, n = 2).

Discussion

In this study, we examined the value of DBS in detecting anti-HCV and quantifying and genotyping HCV RNA using automated and standardized commercial serological and molecular assays.

Anti-HCV and HCV RNA detection was assessed on Whatman 903 cards, which are affordable and readily available worldwide. Our results demonstrate an excellent specificity and sensitivity of DBS anti-HCV detection. Only one false positive result was observed in a single DBS specimen stored at −20°C; however, the confirmatory DBS test was negative, demonstrating the absence of HCV antibodies in this patient. One false negative result was also observed in a sample collected from a human immunodeficiency virus–infected patient with low levels of anti-HCV and undetectable serum HCV RNA. Immunodeficiency is recognized as the commonest cause of false negative results for anti-HCV detection, even in patients with chronic HCV infection.22–24 Our study indicates that the manufacturer's cutoff value for anti-HCV detection in serum applies to the DBS adapted assay. With a sensitivity and specificity close to 100%, anti-HCV detection by way of ELISA in DBS seems appropriate for epidemiological studies, and diagnosis in hard-to-reach populations. Another report that used a modified Monolisa anti-HCV (Sanofi Pasteur, France) technique showed similar results.25

DBS samples with anti-HCV detectable by ELISA may require confirmation by immunoblot, which can also be reliably performed on DBS. Despite the lower quantities of anti-HCV, as a result of the smaller volume of blood in the DBS, we observed similar results to the serum tests, with no indeterminate immunoblot results.

Our study is the first to evaluate HCV RNA quantification on DBS using real-time PCR, automated methods, and commercially available assays. Abe and Konomi have previously reported successful HCV RNA detection on dried serum spots by an in-house, nested PCR in eight patients.26 The use of commercial kits for the extraction method and molecular assays in our study has several advantages: the expertise for assay development and optimization is not required, and quality control can use the manufacturer's control reagents.

We observed a very good concordance between the HCV RNA detected in DBS and serum. The overall sensitivity when using DBS was lower than that for serum in patients with HCV RNA <1,000 IU/mL, but this might have been improved by extracting RNA from larger DBS samples. In our experience, values below 1,000 HCV RNA IU/mL are very uncommon in untreated patients, and therefore this level of sensitivity may suit pretreatment molecular HCV diagnosis. In particular, after controlling for the observed proportional bias, the linear regression analysis of HCV RNA measurements showed a good correlation between the DBS and serum samples, allowing serum HCV RNA quantification from the DBS.

For optimal HCV RNA recovery from Whatman 903 paper, DBS should be frozen at −20°C within 48 hours of collection and drying. Our observations suggest that prolonged exposure at room temperature impairs the HCV RNA recovery, whereas recovery was preserved for frozen DBS. Similarly, Abe and Konomi26 have observed a 10-fold reduction in HCV RNA titers in dried serum stored at room temperature for 4 weeks. In contrast to previous reports,16, 25, 27 we observed a dramatic decrease in assay specificity when testing DBS for anti-HCV after storage at room temperature. Transport of DBS within 2 days after collection is feasible without refrigeration. After this time limit or for remote areas, transport on dry ice may be useful.

DBS is a reliable alternative to serum collection for HCV testing. We showed that 150 μL of whole blood (three drops) spotted onto filter paper allowed the whole panel of HCV testing: anti-HCV detection and confirmation, HCV RNA quantification, and genotyping. DBS sampling may facilitate epidemiological studies on HCV infection and improve the access of high-risk populations to HCV diagnosis and treatment.

Acknowledgements

We thank Abigail King and Emily Witty for editorial assistance in the preparation of this manuscript and Martine Savy and Virginie Zurek for technical assistance

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