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Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 230--235

Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper

Ritapa Ghosh1, Naba Kumar Hazarika2,  
1 Vadodara Institute of Neurological Sciences, Vadodara, Gujarat; Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India
2 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati; Lab Operations, HLL Hindlabs Diagnostic Centre, Assam, India

Correspondence Address:
Dr. Ritapa Ghosh
Vadodara Institute of Neurological Sciences, Vadodara - 390 007, Gujarat
India

Abstract

Purpose: Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA. Materials and Methods: At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjected to anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design. Results: Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781–0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively. Conclusions: DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.

How to cite this article:
Ghosh R, Hazarika NK. Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper.Indian J Med Microbiol 2018;36:230-235

How to cite this URL:
Ghosh R, Hazarika NK. Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper. Indian J Med Microbiol [serial online] 2018 [cited 2019 Dec 12 ];36:230-235
Available from: http://www.ijmm.org/text.asp?2018/36/2/230/238677

Full Text

 Introduction



Hepatitis C virus (HCV) was identified in the late 1980s by the direct molecular approach.[1] Early diagnosis of HCV infection is rare as acute infection is usually asymptomatic. Screening for anti-HCV antibodies with a serological test identifies people who have been infected with the virus. If the test is positive for anti-HCV antibodies, a nucleic acid test for HCV RNA is needed to confirm chronic HCV infection. About 130–150 million people have chronic HCV infection globally and a sizeable number of these develop liver cirrhosis or hepatocellular carcinoma.[2] In India, about 12–13 million HCV carriers have been identified.[3] The prevalence of HCV infection in sex workers, homeless, prisoners and institutionalised individuals is more than the general population. HCV testing in these groups is limited by the poor acceptability or feasibility of venepuncture. Thus, developing countries face considerable problems in serological screening and confirmation of HCV infection.[4]

Dried blood spot (DBS) specimen is a drop of capillary whole blood collected on a filter paper from a simple prick of the finger. 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] We wanted to see if DBS using a cheap filter paper is as reliable as serum specimen for detection of anti-HCV and HCV RNA.

 Materials and Methods



We conducted a study at a tertiary care hospital of Northeast India, from July 2014 to June 2015 with the following objectives: (1) to detect anti-HCV by enzyme-linked immunosorbent assay (ELISA) in the DBS specimens of patients at risk for HCV infection, (2) to confirm HCV infection in ELISA positive specimens with polymerase chain reaction (PCR) and (3) to assess reliability of the DBS specimens against the serum specimens for detection of anti-HCV by ELISA.

Study design

In this observational study, we followed a paired screen-positive design where PCR was done in only those patients who had anti-HCV antibodies by ELISA in the DBS or the serum specimen or both [Figure 1].[5] Performing PCR in all the patients was not feasible due to cost limitations. The study was approved by the Institutional Ethical Committee of our hospital.{Figure 1}

Selection of subjects

We visited the Departments of Medicine, Paediatrics, Gastroenterology and Nephrology of our hospital by turn twice a week to select the patients for the study. Particulars of patients were obtained in a pre-designed pro forma and included demographic features, clinical features, laboratory data and diagnosis.

Inclusion criteria

We selected the patients at risk of HCV infection. These included injection drug users (IDUs), patients with chronic kidney disease on frequent haemodialysis, patients with hepatitis and history of blood transfusion, children born to mothers infected with HCV, sexual partners of HCV infected patients and patients with tattoos or piercings.

Exclusion criteria

We excluded the patients who not at high risk of HCV infection, the patients screened for HCV infection previously and known patients of HCV infection under follow-up.

Collection of specimens

Informed consent was taken for collection of blood specimens. Paired serum and DBS samples were collected from the selected patients. For collection of serum specimens, 5 ml of venous blood was collected by venepuncture in BD vacutainer tubes. One aliquot of blood was centrifuged at 3000 rpm for 10 min to obtain the serum. A properly labelled Whatman No. 1 filter paper (Cat No. 1001 125 from GE Healthcare) was used for collection of DBS specimen. Four 12-mm circles were drawn. After preparing the skin, finger was pricked by single-use lancet following which blood was dropped on each of the four circles.[6]

Storage of specimens

Serum specimens were stored at -70°C. DBS filter papers were air-dried for 4 h at room temperature. Then, they were placed in sealable plastic bags-containing silica desiccant sachets and stored at -20°C.[6],[7]

Detection of anti-hepatitis C virus

Paired serum and DBS eluates were subjected to third-generation anti-HCV ELISA using HCV Microlisa (J. Mitra and Co. Pvt. Ltd.). For DBS elution,

6-mm discs were punched from each blood-soaked circle. The discs were placed in a microcentrifuge tube with 200 μl sample diluent provided in the HCV Microlisa Kit. They were incubated overnight at room temperature in a laboratory rotator. DBS eluate and serum were tested for anti-HCV on the next day. The DBS eluate was tested with a slight modification. We used 100 μL of DBS eluate. All the subsequent steps including interpretation were followed according to manufacturer's instructions. The cut-off optical density (OD) value was calculated as the mean OD of positive controls X 0.23 (as per the manufacturer's guideline).

Detection of hepatitis C virus RNA by polymerase chain reaction

Confirmation of HCV infection in ELISA positive serum and DBS samples was done by real-time reverse transcriptase PCR (RT-PCR). HCV RNA was extracted following the manufacturer's instructions from the serum specimen using the 'QIAamp Viral RNA Mini Kit, Qiagen'. We introduced the internal control of PCR (the assay used brome mosaic virus) into each sample and the negative control at the lysis buffer stage of the extraction process. A single step real-time RT-PCR protocol was carried out on the serum samples using the commercially available real-time HCV RNA detection set (Fast track diagnostics) and the assays were run using real-time RT-PCR LightCycler ® 480 II (Roche Diagnostic GmbH, Mannheim, Germany). Results were displayed on a computer using the Light Cycler 480 Software version 1.5 connecting the Light Cycler 480 II instrument. Viral elution from DBS was performed using two 6-mm spots cut from the 12-mm pre-drawn circle by a puncher. The pieces were suspended in a 1.5 mL Eppendorf 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 overnight. After centrifugation (20 s at 13,000 g), the supernatant was collected for extraction.[4] All the subsequent steps were same as in the serum.

Statistical analysis

Using statistical package for the social sciences (SPSS Statistics for Windows, version 17.0, released 2008, SPSS Inc., Chicago, USA), the means and standard deviations (SDs) of the ELISA OD values were calculated. Comparison of the means was done by Mann-Whitney test. Pearson's correlation coefficient was calculated to study the correlation between the ELISA OD values of the serum and the DBS specimens and to study the correlation between HCV RNA levels in the serum and the DBS specimens. P < 0.05 was considered statistically significant. The measures of accuracy of DBS ELISA were calculated in the form of relative sensitivity rate and relative false-positive rate.

 Results



A total of 91 patients were selected during the study period. The median age of these patients was 43 years (range, 6–76 years). Male-to-female ratio was 3.8: 1. The clinical features of these patients were variable and included pallor, anorexia, fatigue, jaundice, fever, abdominal pain, nausea, vomiting, upper gastrointestinal bleed, weight loss, pedal oedema, hepatomegaly, splenomegaly, ascites and altered sensorium. All the selected patients had risk factors for HCV infection [Table 1].{Table 1}

Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens [Figure 2]. There was discordance in the detection of anti-HCV in one paired DBS and serum specimen [Table 2]. Calculated cutoff OD values for DBS and serum specimens were 0.564 and 0.595, respectively. The mean OD values of the reactive DBS and serum specimens were 1.758 (SD, 0.555) and 1.490 (SD, 0.877), respectively. There was no significant difference in the OD values of the positive paired DBS and serum specimens (P = 0.6606). While analysing the OD values of all the DBS and serum specimens, Pearson correlation coefficient (r) was found to be 0.9181 (95% confidence interval, 0.8781–0.9453). Coefficient of determination (r2) was 0.8428 (P < 0.0001). This suggested a strong correlation between the OD values of anti-HCV ELISA in DBS and serum samples [Figure 3].{Figure 2}{Table 2}{Figure 3}

HCV RNA was detectable in 5/9 (55.6%) DBS specimens reactive for anti-HCV and 9/10 (90.0%) serum specimens reactive for anti-HCV [Table 2]. There was no correlation between the HCV RNA levels in the serum and DBS samples (P = 0.9512). Pearson's correlation coefficient (r) was −0.0223 (95% confidence interval, −0.6430–0.6161). Coefficient of determination (r2) was 0.0004 [Figure 3].

Performing the gold standard test for chronic HCV infection (HCV RNA by RT-PCR) in all the specimens was not feasible due to cost limitations. Therefore, the sensitivity and specificity of anti-HCV ELISA on the DBS specimen could not be estimated. In the paired-screen positive design that we followed, the measures of accuracy are the relative sensitivity rate and the relative false-positive rate.[5] The calculated relative sensitivity rate of the DBS anti-HCV ELISA was 0.89. The calculated relative false-positive rate of the DBS anti-HCV ELISA was 1.00 [Table 3]. These results are interpreted as follows. Anti-HCV ELISA on the DBS specimen was 89% as sensitive as anti-HCV ELISA on the serum specimen. The DBS specimen was as much as likely as the serum specimen to give a false-positive anti-HCV ELISA result. Thus, anti-HCV ELISA on the DBS specimen was as specific as anti-HCV ELISA on the serum specimen.{Table 3}

 Discussion



DBS technology has applications in neonatal metabolic screening, therapeutic drug monitoring, preclinical and clinical pharmacokinetics, toxicokinetic, forensic, biological and immunological sciences. In the field of microbiology, it has been used to study the prevalence of various viral, bacterial and parasitic infections. Most studies on detection of anti-HCV in DBS [Table 4] were conducted with good quality filter papers approved by the United States (US) FDA (US Ahlstrom 226-K062932, Whatman 903 and PerkinElmer 226).[8],[9] To the best of our knowledge, only one study (from Lahore, Pakistan) used Whatman No. 1 filter paper for detection of anti-HCV in patients (women of childbearing age and children). The DBS specimens were screened by an enzyme immunoassay (Chiron Corporation, Emeryville, Calif.). Further tests on the positive specimens were included test based on a gelatin particle agglutination assay (Fujirebio Inc., Tokyo, Japan) and confirmation by a third-generation immunoblot assay (RIBA 3.0; Ortho-Clinical Diagnostics, Amersham, United Kingdom). Serum specimens were not obtained.[14] Whatman No. 1 filter paper is available at a cheap price. We wished to determine if DBS using this filter paper is as reliable as serum specimen in detecting anti-HCV by ELISA commonly used in India (HCV Microlisa, J. Mitra and Co. Pvt. Ltd.). In addition, we wanted to know if it is a reliable specimen for detection of HCV RNA by PCR.{Table 4}

There was good correlation between the OD values of anti-HCV ELISA in the DBS and the serum specimens (r = 0.9181, P < 0.0001) in our study. DBS anti-HCV ELISA was as specific as and 89% as sensitive as serum anti-HCV ELISA. Another study from Chennai, India, had also found good correlation (r = 0.98) between the OD values of anti-HCV ELISA in the DBS and the serum specimens.[10] The strength of correlation between anti-HCV titres of DBS and serum specimens was lower (r = 0.631) in a study from Malaysia.[12] The authors had used Whatman 903 for collecting DBS specimen in these studies.

The lower sensitivity of DBS anti-HCV ELISA in our study compared to others could be due to several factors such as different type of filter card, different assay kit and longer storage duration in our study. Our study had one false-negative result in a DBS specimen. The OD values of anti-HCV ELISA in the DBS and serum specimens were 0.033 and 0.896, respectively. HCV RNA was not detectable in the DBS specimen, but the serum HCV RNA load was 2630 IU/mL. The false-negative result in the DBS specimen could possibly be due errors in DBS sample collection and elution. There was one false-positive result with anti-HCV ELISA in both DBS and serum specimens. The OD values of DBS and serum specimens were 1.328 and 1.055, respectively. HCV RNA was not detectable in either of DBS or serum specimen. The patient's serum was reactive to HBsAg. The likely reason of false-positive test could be resolved or past HCV infection, transient low viremia with episodic viral replication or suppression of HCV by HBV.[11],[15]

Some authors have demonstrated high HCV RNA detection rate in the DBS specimens and good correlation between the HCV RNA quantity in the DBS and the serum specimens.[4] However, many authors believe that the correlation is imprecise and rely on the DBS specimen only for a qualitative determination of the presence or absence of HCV RNA in blood.[16],[17],[18] In our study, HCV RNA was detectable in only 55.6% of the DBS specimens reactive for anti-HCV. There was no correlation between HCV RNA levels in the DBS and serum specimens (r = −0.0223). Whatman No. 1 filter paper is not intended for conservation of RNA. RNA is notoriously unstable and continuous RNA degradation during storage has been reported previously.[19] A standard 1-cm-diameter DBS punch size holding 50 μL blood is less efficient for nucleic acid extraction.[20] In addition, our patients had lower mean serum HCV RNA levels. These might have been the reasons for lower HCV RNA detection rates in the DBS specimens in our study.

About 11% of the patients included in the study had confirmed chronic HCV infection. The patients had been selected based on the presence of risk factors for having HCV infection. Assessment of the risk factors showed that only IDU had a significant association with HCV infection (P < 0.0027).

 Conclusions



Multiple studies have shown that the DBS may be a reliable alternative specimen to serum for detection of anti-HCV and HCV RNA. The benefits of DBS include simplification of sample collection, processing, storage and shipment and increased opportunities of screening for HCV infection in rural, remote and hard-to-reach regions.[4],[10] The limitation of our study is that we followed a paired screen-positive design and all the samples could not be tested for HCV RNA. However, we showed that a cheap filter paper like Whatman No. 1 may be used for collecting DBS specimen. It may not be a suitable specimen for confirmation of HCV infection using PCR, but it is quite reliable as a serum specimen for screening of HCV infection. It can be useful in effective surveillance and field level research in resource-limited settings.

Acknowledgement

We are grateful to the Department of Biotechnology of Tezpur University, Assam for funding this study.

Financial support and sponsorship

Department of Biotechnology, Tezpur University, Assam.

Conflicts of interest

There are no conflicts of interest.

References

1Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-62.
2WHO. Hepatitis C Fact Sheet; July, 2015. Available from: http://www.who.int/mediacentre/factsheets/fs164/en/. [Last accessed on 2015 Aug 30].
3Narahari S, Juwle A, Basak S, Saranath D. Prevalence and geographic distribution of hepatitis C virus genotypes in Indian patient cohort. Infect Genet Evol 2009;9:643-5.
4Tuaillon E, Mondain AM, Meroueh F, Ottomani L, Picot MC, Nagot N, et al. Dried blood spot for hepatitis C virus serology and molecular testing. Hepatology 2010;51:752-8.
5Cheng H, Macaluso M. Comparison of the accuracy of two tests with a confirmatory procedure limited to positive results. Epidemiology 1997;8:104-6.
6Grüner N, Stambouli O, Ross RS. Dried blood spots – Preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp 2015;(97):52619.
7Bertagnolio S, Parkin NT, Jordan M, Brooks J, García-Lerma JG. Dried blood spots for HIV-1 drug resistance and viral load testing: A review of current knowledge and WHO efforts for global HIV drug resistance surveillance. AIDS Rev 2010;12:195-208.
8Sharma A, Jaiswal S, Shukla M, Lal J. Dried blood spots: Concepts, present status, and future perspectives in bioanalysis. Drug Test Anal 2014;6:399-414.
9Smit PW, Elliott I, Peeling RW, Mabey D, Newton PN. An overview of the clinical use of filter paper in the diagnosis of tropical diseases. Am J Trop Med Hyg 2014;90:195-210.
10Nandagopal P, Iqbal HS, Saravanan S, Solomon SS, Mehta S, Selvakumar M, et al. Evaluation of dried blood spot as an alternative specimen for the diagnosis of anti-HCV in resource-limited settings. Indian J Med Microbiol 2014;32:208-10.
11Ross RS, Stambouli O, Grüner N, Marcus U, Cai W, Zhang W, et al. Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots – Performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification. Virol J 2013;10:72.
12Lee CE, Sri Ponnampalavanar S, Syed Omar SF, Mahadeva S, Ong LY, Kamarulzaman A, et al. Evaluation of the dried blood spot (DBS) collection method as a tool for detection of HIV Ag/Ab, HBsAg, anti-HBs and anti-HCV in a Malaysian tertiary referral hospital. Ann Acad Med Singapore 2011;40:448-53.
13Croom HA, Richards KM, Best SJ, Francis BH, Johnson EI, Dax EM, et al. Commercial enzyme immunoassay adapted for the detection of antibodies to hepatitis C virus in dried blood spots. J Clin Virol 2006;36:68-71.
14Parker SP, Khan HI, Cubitt WD. Detection of antibodies to hepatitis C virus in dried blood spot samples from mothers and their offspring in Lahore, Pakistan. J Clin Microbiol 1999;37:2061-3.
15Zarski JP, Bohn B, Bastie A, Pawlotsky JM, Baud M, Bost-Bezeaux F, et al. Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 1998;28:27-33.
16McDade TW, Williams S, Snodgrass JJ. What a drop can do: Dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 2007;44:899-925.
17De Crignis E, Re MC, Cimatti L, Zecchi L, Gibellini D. HIV-1 and HCV detection in dried blood spots by SYBR green multiplex real-time RT-PCR. J Virol Methods 2010;165:51-6.
18Santos C, Reis A, Dos Santos CV, Damas C, Silva MH, Viana MV, et al. The use of real-time PCR to detect hepatitis C virus RNA in dried blood spots from Brazilian patients infected chronically. J Virol Methods 2012;179:17-20.
19Fiscus SA, Brambilla D, Grosso L, Schock J, Cronin M. Quantitation of human immunodeficiency virus type 1 RNA in plasma by using blood dried on filter paper. J Clin Microbiol 1998;36:258-60.
20Greenman J, Roberts T, Cohn J, Messac L. Dried blood spot in the genotyping, quantification and storage of HCV RNA: A systematic literature review. J Viral Hepat 2015;22:353-61.