|Year : 2019 | Volume
| Issue : 1 | Page : 60-66
Evaluation of dried blood spots as a feasible alternative to plasma for the detection and quantification of hepatitis c virus in a tropical setting: A pilot study
Jai Ranjan1, Suresh Ponnuvel1, Gnanadurai John Fletcher1, Raghavendran Anantharam1, Kalaivani Radhakrishnan1, Visalakshi Jeyaseelan2, Priya Abraham1
1 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Web Publication||16-Aug-2019|
Dr. Priya Abraham
Department of Clinical Virology, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Introduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients' blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log10IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.
Keywords: Directly acting antivirals, dried blood spots, hepatitis C virus
|How to cite this article:|
Ranjan J, Ponnuvel S, Fletcher GJ, Anantharam R, Radhakrishnan K, Jeyaseelan V, Abraham P. Evaluation of dried blood spots as a feasible alternative to plasma for the detection and quantification of hepatitis c virus in a tropical setting: A pilot study. Indian J Med Microbiol 2019;37:60-6
|How to cite this URL:|
Ranjan J, Ponnuvel S, Fletcher GJ, Anantharam R, Radhakrishnan K, Jeyaseelan V, Abraham P. Evaluation of dried blood spots as a feasible alternative to plasma for the detection and quantification of hepatitis c virus in a tropical setting: A pilot study. Indian J Med Microbiol [serial online] 2019 [cited 2019 Oct 21];37:60-6. Available from: http://www.ijmm.org/text.asp?2019/37/1/60/264485
| ~ Introduction|| |
Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus of 9.6 kb belonging to family Flaviviridae and genus Hepacivirus. The global prevalence of HCV is about 1%, with India having a prevalence of 0.09%–2.02%, with about 6–11 million people infected. HCV is a major cause of morbidity and mortality, with only about 15%–25% of acute infections leading to resolution and the rest progressing to chronicity.
HCV infection can be diagnosed by rapid diagnostic tests (RDTs) and serological tests such as enzyme-linked immunosorbent assays and chemiluminescent micro-particle immunoassay (CMIA), which mainly detect the antibody response to such an infection. Serological tests, particularly RDTs are performed in low-resource settings because of their wider availability and ease of use. Molecular tests that detect and quantify HCV RNA are the gold standard, which if positive imply an active infection. They also help in monitoring response to therapy as the viral RNA decreases with effective therapy.
There are seven confirmed genotypes of HCV, but in recent years, genotyping of HCV has become essential only for epidemiological purposes. Recently, HCV core antigen detection has also been employed as an alternative to HCV RNA quantification as it rises early in the infection and correlates well with HCV RNA levels, giving a better idea of level of replication of the virus or its decline.
With the advent of directly acting antivirals (DAAs), there is a possibility of cure. DAAs are cost-effective, efficacious medications with fewer side effects which should become available to all individuals with demonstrable HCV viraemia even in the remote, less accessible areas of the country.
Many people living in remote areas are still not aware that they have HCV infection and there is an urgent need for better sampling and transportation of samples from such distant areas to reference centres, where the facility for the diagnosis of HCV is available. This has become even more important with the recent action plan of the WHO to eliminate hepatitis C by 2030.
Detection of HCV RNA is required to start the treatment and assess the response. Collection of plasma and its appropriate transport to referral laboratories from distant resource-limited settings is challenging and may negatively impact on the accuracy of results. The challenges are due to the duration of shipment and extremes of temperature that the clinical samples can be subjected to.
Dried blood spots (DBS) have recently been evaluated as an alternative to plasma for detecting HCV RNA and HCV genotyping, and it has been recommended in the recent WHO guidelines.
There are not many studies evaluating DBS as an alternative sample for HCV RNA detection in our country. In addition, the influence of extreme ambient temperatures of the Indian subcontinent on such samples during shipment has not been addressed to.
Aim and objectives
The aim of this study is to assess the feasibility of DBS as an alternative specimen to plasma for HCV RNA detection and quantification.
- To compare DBS with plasma as an alternative specimen source to detect/quantify HCV RNA and hepatitis C core antigen
- To analyse the effect of different temperatures (4°C and ≥37°C for 15 days) on DBS samples, before detection and quantification of HCV RNA, and HCV core antigen.
| ~ Methods|| |
This study was approved by the Institutional Review Board and was conducted from March 2016 to May 2017.
Samples from 40 consecutive treatment-naive patients, with high index of suspicion of having HCV infection; referred from the liver clinic were included in the study after written informed consent. Patients with HCV RNA-negative plasma, patients who were pregnant or <18 years of age and who did not give consent for the study were excluded. Patient demographics and liver function test results were collected from the electronic clinical records.
Nine millilitres of blood was collected in sterile vacutainer tubes-containing potassium ethylene diamine tetraacetic acid (K2 EDTA). An additional 4 ml K2 EDTA tube was taken, of which 2 ml blood was used for estimating packed cell volume (PCV). This extra sample was collected because 6–7 ml of whole blood (after separating out plasma) is used for routine testing, whereas the remaining 1–2 ml of whole blood was used for generating DBS. PCV values were estimated for each sample, to arrive at a correction factor/normalisation coefficient. This was to adjust for the difference in sample volume and sample type between plasma and DBS samples.
Two Whatman 903™ (GE Healthcare, Cardiff, UK) protein saver cards were labelled with unique study identification number, date of collection and the temperature (4°C or ≥37°C) and whole blood was spotted onto them. Each Whatman 903™ card has five sample slots with each having a diameter of about half inches. Fifty microlitres of whole blood was spotted onto each of the sample slots in two cards for each patient (Totally 500 μl spotted, on two cards). The cards were labelled and left to dry, at room temperature (28°C ± 3°C) for 6 h. Cards were then put into separate zip lock pouches with desiccant. Afterwards, the card labelled as DBS A (4°C) was placed in a box and stored in refrigerator which maintained 4°C and the card labelled as DBS B (≥37°C) was put in another box and kept in walk in incubator with temperature ≥37°C (maximum temp: 39°C), for 15 days. After 15 days, the cards were taken out of their respective boxes and kept at −20°C, until the time of testing.
Two sample slots from each DBS card (A and B), containing 50 μl individually were punched out and used for elution. HCV RNA elution was performed according to a modification of the protocol by David et al. using Abbott bulk m Lysis buffer. Two sample slots from each DBS card were punched out (three punches from one slot) and placed into 1.7 ml of m Lysis buffer aliquoted into Tarsons tubes. These were kept at room temperature (28°C ± 3°C) for 2 h with intermittent agitation. After 2 h, Tarsons tube with the DBS punches were centrifuged at 4500 rpm for 2 min. Eluate (750 μl) was then used for HCV-RNA extraction by Abbott M2000 sp. The RNA extract was subjected to nucleic acid amplification using Abbott RealTime HCV assay (Abbott Molecular Inc., Des Plaines, IL, USA) which targets 5' untranslated region of HCV genome.
A total of 36 samples were included in the final statistical analysis, as HCV RNA values were available for all three subsets, i.e., plasma, DBS A (at 4°C) and DBS B (at ≥37°C) for 36 of these study patients. Other four samples were excluded, as any one of the subset (either DBS A or DBS B) of these samples gave invalid result or error codes on repeated testing. This was done for better correlation analysis and to minimise any statistical error.
A normalisation coefficient was calculated to adjust for sample type and sample volume difference between DBS samples and plasma samples., HCV is predominantly present in plasma. Since whole blood is spotted on the Whatman 903™, there is a requirement to calculate a normalisation coefficient for estimating the corresponding plasma viral load of each DBS sample. This is due to the difference in plasma volume in the DBS in comparison with routine plasma used for HCV quantification. Median haematocrit (PCV) value for 36 patients included in the study was 37.4% (interquartile range: 30.1–41.05). In DBS, the input volume was 100 μl. Subtracting the median PCV value from hundred will give the amount of plasma in the DBS (100%-37.4% = 62.6%). The correction factor for ascertaining plasma viral load in 100 μl was calculated as, 100/62.6 = 1.6.
However, in routine testing, 500 μl of plasma is used, but in DBS testing, 100 μl of whole blood is spotted and eluted for estimation of HCV RNA. The correction factor adjusting for the volume of sample was 500/100 = 5.
Thus, the final normalisation/correction factor taking into consideration both the volume and sample type was 8 (5 × 1.6).
HCV core antigen estimation was undertaken on 29 paired DBS sample eluates employing the Abbott ARCHITECT HCV Ag Assay (Abbott Laboratories, USA), which is an automated CMIA.
The agreement between routine plasma and corrected DBS based polymerase chain reaction (PCR) was analysed using Interclass correlation. Bland Altman Plot was used to compare the agreement between the two methods. All data generated in the study were analysed using Microsoft Excel and SPSS software (IBM Corp., Armonk; New York, USA).
| ~ Results|| |
Demographic profile and baseline parameters
Of the 36 study patients, 33.3% were from Bangladesh, which formed the single largest ethnic group of this study. Patients from West Bengal (19.4%), Tamil Nadu (13.8%) and Andhra Pradesh (8.3%) were the next significant population subset. The rest of the study participants were from Jharkhand, Bihar, Madhya Pradesh, Sikkim, Assam and Mizoram. The median age of patients in our study was 51 (interquartile range: 42.5–63.5) years.
Liver function and coagulation profiles were obtained from clinical records. The median aspartate aminotransaminase level was 47.5 IU/L (interquartile range: 31.75–88.75). Median alanine aminotransferase and alkaline phosphatase values were 54.5 IU/L (interquartile range: 26.5–77) and 81 IU/L (interquartile range: 67.5–114.5), respectively. Prothrombin time (PT) values were available for 34 patients (median: 11.25 s) and activated partial PT had a median of 37.4 s (for 25 patients).
The mean HCV antibody levels of the thirty six samples included for statistical analysis was 11.97 S/Co. The median HCV antibody was 12.625 S/Co (interquartile range: 10.75–13.825).
Genotype 3 (72.7%) was the predominant genotype followed by genotype 1 (24.2%) and genotype 4 (3.03%) among the study population.
Hepatitis C virus RNA estimation
The mean plasma HCV RNA values and mean plasma HCV RNA log10 values for 36 study patients were 168,2975 IU/mL and 6.22 log10 IU/mL, respectively. Median plasma HCV RNA for 36 patients was 554351 IU/mL (interquartile range: 139,235–2,095,924) and median log10 plasma HCV RNA value (for 36 patients) was found to be 5.74 (interquartile range: 5.14–6.32) log10 IU/mL.
Comparison of plasma hepatitis C virus RNA and dried blood spots hepatitis C virus RNA kept at 4°C (Dried blood spots A)
Median HCV RNA values of DBS samples kept at 4°C was 5708 (interquartile range: 1279–15,127.50) IU/ml. Corresponding median log10 HCV RNA value was 3.75 (interquartile range: 3.10–4.17) log10 IU/mL.
The difference in plasma HCV RNA log10 value and DBS (4°C) HCV RNA log10 value is 1.99 logs.
The normalised median log10 HCV RNA for DBS A (at 4°C) was 4.66 (interquartile range: 3.98–5.09) log10 IU/mL and the difference between median plasma log10 HCV RNA, i.e., 5.74 (interquartile range: 5.14–6.32) and median log10 HCV RNA of DBS A was 1.08 logs.
Plasma HCV RNA values and normalised DBS A, HCV RNA values showed a high level of agreement with Pearson's correlation coefficient (R) of 0.950 (95% confidence interval: 0.903–0.974) and P < 0.0001, as shown in [Figure 1].
|Figure 1: Correlation between normalised dried blood spots A log10hepatitis C virus RNA (4°C) (X-axis) and plasma log10hepatitis C virus RNA (Y-axis), showing high correlation|
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Bland–Altman plot showed a high agreement between plasma HCV RNA values and normalised/corrected DBS A (4°C) HCV RNA values, as depicted in [Figure 2].
|Figure 2: Bland–Altman plot showing correlation between plasma log10hepatitis C virus RNA and normalised dried blood spots A (4°C) log10hepatitis C virus RNA values|
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Comparison of plasma hepatitis C virus RNA and dried blood spots hepatitis C virus RNA kept at ≥37°C(dried blood spots B)
Median HCV RNA value for DBS samples kept at ≥37°C was 9800 (interquartile range: 1625–20,169) IU/ml. Median log10 HCV RNA value for such samples was 3.99 (interquartile range: 3.21–4.30) log10 IU/mL.
The difference between the median plasma log10 HCV RNA values (5.74 log10 IU/mL) and median log10 HCV RNA value for DBS B (3.99 log10 IU/mL) was 1.75 logs.
The normalised median log10 HCV RNA for DBS B (at ≥37°C) was 4.92 (interquartile range: 4.14–5.12) log10 IU/mL. The difference between median plasma log10 HCV RNA and median of corrected/normalised DBS B was 0.82 logs.
Normalised log values of HCV RNA of DBS B (at ≥37°C) and plasma log values of HCV RNA showed a high correlation, with the correlation value; R = 0.943 (95% confidence interval: 0.892–0.971), P < 0.0001, as shown in [Figure 3].
|Figure 3: Correlation between normalised dried blood spots B (≥37°C) log10(X-axis) hepatitis C virus RNA and plasma log10hepatitis C virus RNA (Y-axis)|
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Bland–Altman plot analysing HCV RNA values of plasma and DBS B (at ≥37°C) (corrected/normalised value) showed a high agreement, as depicted in [Figure 4].
|Figure 4: Bland–Altman plot showing correlation between hepatitis C virus RNA log10values of plasma and respective dried blood spots B (≥37°C) samples|
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Comparison of log values of hepatitis C virus RNA of dried blood spots A (at 4°C) and dried blood spots B (at ≥ 37°C)
Good correlation (R = 0.974, 95% confidence interval: 0.950–0.987, P < 0.0001) was found between HCV RNA log10 values of DBS A and B. The median log10 HCV RNA value of DBS A was 3.75 (interquartile range: 3.10–4.17) log10 IU/mL, whereas of DBS B was 3.99 (interquartile range: 3.21–4.30) log10 IU/mL. The corrected/normalised median log10 HCV RNA for DBS A was 4.66 (interquartile range: 3.98–5.09) log10 IU/mL and median log10 HCV RNA for DBS B was 4.92 (interquartile range: 4.14–5.12) log10 IU/mL.
Hepatitis C virus core antigen estimation
Plasma hepatitis C virus RNA and plasma hepatitis C virus core antigen comparison
Plasma HCV core antigen values were available for 34 plasma samples. These values were compared with plasma HCV RNA log10 values of corresponding samples.
Median plasma HCV RNA log10 value for the 34 samples taken into consideration was 5.73 (interquartile range: 4.99–6.23) log10 IU/ml and median plasma log10 HCV core antigen values for the same plasma samples was 2.70 (interquartile range: 1.89–3.48) log10 fmol/L. The scatter plot showed correlation, i.e., R = 0.777 (95% confidence interval: 0.591–0.884), P < 0.0001.
Plasma hepatitis C virus core antigen and dried blood spots (A and B) hepatitis C virus core antigen results comparison
DBS (A and B) HCV core antigen values were available for 29 samples, and hence, these were compared with corresponding plasma HCV core antigen values for a better comparison.
Median core antigen value for 29 plasma samples was 325.35 fmol/L.
Median HCV core antigen values for DBS A and DBS B were found to be 4.64 (interquartile range: 3.73–5.63) fmol/L and 4.77 (interquartile range: 3.77–6.14) fmol/L, respectively.
Comparison of hepatitis C virus core antigen values of plasma and dried blood spots A (at 4°C)
Poor correlation (R = 0.031, 95% confidence interval: −0.33–0.39) was found between core antigen values of plasma and DBS A samples.
Comparison of hepatitis C virus core antigen values of plasma and dried blood spots B (at ≥37°C)
Poor correlation (R = 0.164, 95% Confidence interval: −0.22–0.506) was found between core antigen values of plasma and DBS B (at ≥37°C).
| ~ Discussion|| |
Patient care of HCV encompasses screening and timely diagnosis of the disease and monitoring the patients through the course of therapy to monitor the treatment response. HCV RNA quantification is considered the best method to assess therapy as antibodies to HCV remains positive even in individuals who have spontaneously cleared the virus. HCV RNA is also superior to antibody detection in immunosuppressed patients, where HCV antibody maybe falsely negative and in individuals with high-risk lifestyles and in those who are multi-transfused, as they have high chances of getting re-infected.
HCV RNA detection is available only in few specialised centres in a country such as India. In the absence of such tests in distant resource-limited settings, samples are sent to referral centres for HCV RNA quantification.
DBS have been used in the recent past as a means of easy sampling of patients and for convenient storage and transport of samples from distant resource-limited settings to referral laboratories for testing of HCV. Effect of storage conditions, ambient room temperature and duration of storage on HCV RNA in DBS has also been evaluated in different studies in other regions.
An earlier study evaluating the stability of HCV RNA in DBS by Abe and Konomi showed that HCV RNA remained stable at room temperature for 4 weeks, but there was a 10-fold decline in the viral load over the period. Solmone et al. assessed HCV RNA from a set of DBS samples stored at room temperature for 11 months and found that the positivity of the samples was preserved throughout the study. Tuaillon et al. (2010), in their study, found dramatic decrease in HCV RNA in DBS samples kept at room temperature for 6 days, while DBS kept at −20°C showed no significant decline. HCV RNA was found to be stable at different temperatures for a period of 1 year in the study by Bennett et al. In the study published in 2013, Brandão et al. showed DBS to be stable at different conditions such as 22°C–26°C, 2°C–8°C and −20°C for 60 days. Cloherty et al., in their study, found HCV RNA to be stable in DBS for 10 weeks at ambient room temperature and at 2°C–8°C. No substantial difference was observed in HCV RNA DBS samples stored at 24°C and at −80°C over 19 months in a study by Soulier et al. Neesgaard et al., from Denmark compared plasma HCV RNA values and DBS HCV RNA values of samples sent through regular mail and found a statistically significant correlation (R = 0.678) between both.
Majority of the studies have evaluated the effect of ambient room temperature on DBS in temperate climates and did not assess the effect of tropical temperatures on the stability of HCV RNA. Recent Indian studies by Mahajan et al. and Ghosh andHazarika have evaluated DBS as a means of sample collection, but there are limited data on the effect of elevated temperatures prevalent in the Indian subcontinent on the integrity of HCV RNA.
In this study, we have tried to simulate temperature conditions during transport from distant sites in the tropical climate widely present in India by storing DBS cards at ≥37°C for 15 days. DBS cards were also stored at 4°C to simulate the transport of such cards in a controlled cooler environment. These cards were then stored at −20°C until time of testing, in the same run. Plasma from the same patient was subjected to routine viral load testing. HCV RNA quantification and HCV core antigen estimation of DBS was undertaken to evaluate the feasibility of DBS as a specimen, by comparing DBS results with the corresponding plasma results.
In this study, median log plasma HCV RNA value was 5.74 (interquartile range: 5.14–6.32) log10 IU/mL. Median log values of HCV RNA in DBS A and DBS B were 3.75 (interquartile range: 3.10–4.17) log10 IU/mL and 3.99 (interquartile range: 3.21–4.30) log10 IU/mL, respectively.
A difference of 1.99 log10 IU/mL was observed between median log value of plasma HCV RNA and median log HCV RNA value of DBS A (4°C). DBS B (at ≥37°C) showed a difference of 1.75 log10 IU/mL. This was in accordance with results from previously published studies by Marins et al., which showed 2 log10 IU/mL difference in plasma log10 HCV RNA and DBS log10 HCV RNA values and a study in France undertaken by Soulier et al. where mean HCV RNA level of DBS with whole blood was 1.75 ± 0.3 log10 IU/mL less than that of plasma. This signifies that DBS can be used as means of sampling for HCV RNA as the difference between plasma and DBS HCV RNA log10 values is consistent in different studies and DBS HCV RNA values can be utilised to predict the plasma HCV RNA value.
There was strong correlation between HCV RNA log10 values of, DBS A (R = 0.950, P < 0.0001) and DBS B HCV RNA log10 values (R = 0.943, P < 0.0001) with that of the plasma log10 HCV RNA value. This again is in concordance with the published data., Results from this study suggest that no deterioration of HCV RNA occurred at high-temperature conditions (≥37°C). Thus, DBS can be used as a means to transport samples even in the Indian subcontinent where the temperatures are high, especially during the summer.
Using Bland–Altman analysis, a high agreement between the values of the DBS log10 HCV RNA and plasma log10 HCV RNA was demonstrated in this study, with 95% of values should lying between −1.96 standard deviation [SD] and +1.96 SD.
Whole blood is spotted onto DBS while plasma is the preferred sample for testing of HCV RNA. There is a difference in sample volume and sample type between plasma and DBS samples, which need to be adjusted for giving an accurate result. PCV values of samples are required for arriving at a correction factor, as is shown in studies by David et al. and Marins et al.
In our study, DBS samples were normalised with respect to sample type and volume, by the use of a correction factor/normalisation coefficient (ascertained by median PCV value of the study participants, 37.4%) and showed a reduction of 0.82–1.08 log10 IU/mL from that of plasma HCV RNA values, suggesting that there is some amount of loss in efficiency of amplification in using DBS samples, again in accordance with the finding in the study by Marins et al.
Sample with plasma HCV RNA value of 542 IU/mL (2.73 log10 IU/mL) was detected in both DBS A and B. DBS A yielded a result of '<12 IU/mL detected,' whereas DBS B yielded a result of 26 IU/ml (1.46 log10 IU/mL).
Another sample with plasma HCV RNA of 17 IU/ml (1.23 log10 IU/mL) was not detected in both of the corresponding A and B DBS cards, showing the lower limit of detection of HCV RNA by DBS in our study to be around 542 IU/ml (2.73 log10 IU/ml).
There have been a few studies which have assessed the lower limit of detection of HCV RNA by DBS. Tuaillon et al. found the lowest limit of detection of HCV RNA by DBS to be 331 IU/mL. Bennett et al. concluded that for the HCV RNA to be detected in DBS, the value in the corresponding plasma sample should be between 150 and 250 IU/mL. Marques et al. and Mössner et al. found the lower detection limit of HCV RNA to be 58 copies/mL and <100 IU/mL, respectively., Furthermore, in a recent Indian study, 3 log10 IU/mL was considered to be the lowest HCV RNA viral load that was detected by employing DBS. The lower limit of detection of HCV RNA in our study is higher than that found in other published studies. A study with larger sample size is required to ascertain the limit of detection of HCV RNA in DBS samples accurately.
HCV core antigen is a structural protein of HCV which is detected in blood during viral replication. Its detection in blood is a cost-effective method to diagnose active HCV infection, as HCV RNA PCR is a costly and cumbersome procedure. It is present in blood during the preseroconversion period and can also be used as a marker of infection in neonates and immunocompromised individuals in whom antibody response is not reliable for diagnosis.
As HCV core antigen estimation is easier and requires less equipment than HCV RNA analysis and still gives a clear indication about the infection, HCV core antigen estimation has the potential of being used as a marker for screening and treatment response in low-resource areas. HCV core antigen was also estimated in a total of 29 samples. DBS A and B and plasma HCV core antigen values were poorly correlated (A, 4°C, r = 0.31; B, ≥37°C, r = 0.16). This can be due to the prolonged storage of DBS samples at different temperatures for 15 days, which might have led to deterioration of core antigen. HCV core antigen estimation was undertaken on DBS samples by Brandão et al. and they showed a deterioration in the OD values after storage of DBS samples at −20°C. Soulier et al. also found that DBS had poor sensitivity for quantifying HCV core antigen. Thus, our findings were corroborated.
| ~ Conclusion|| |
This study has demonstrated that DBS can be used as an alternative to plasma for qualitative identification of HCV infection in resource-limited settings. The difference in DBS log10 values and plasma log10 values is in concordance with previous studies. A normalisation/correction factor needs to be deduced while dealing with DBS samples, as the sample volume in DBS is less than that of plasma used for testing and the fact that whole blood is spotted onto DBS which contains blood cells in addition to plasma. The uniqueness of this study is that it shows there is no significant deterioration of nucleic acids in samples kept at ≥37°C compared to those stored at 4°C. Thus, DBS shows promise as a means of sample collection in far-flung resource-limited settings, even in tropical countries. The limitation of this study was the small sample size. A larger study is required for predicting the feasibility of DBS samples as an alternative to plasma, primarily for estimation of HCV core antigen.
Financial support and sponsorship
Fluid Research Grant, Christian Medical College, Vellore. (IRB Min No: 9687 [DIAGNO] dated 20.10.2015) and Virology Special Fund, Department of Clinical Virology, Christian Medical College, Vellore.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Blach S, Zeuzem S, Manns M, Altraif I, Duberg AS, Muljono DH, et al
. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: A modelling study. Lancet Gastroenterol Hepatol 2017;2:161-76.
Colin C, Lanoir D, Touzet S, Meyaud-Kraemer L, Bailly F, Trepo C, et al.
Sensitivity and specificity of third-generation hepatitis C virus antibody detection assays: An analysis of the literature. J Viral Hepat 2001;8:87-95.
Welsch C, Jesudian A, Zeuzem S, Jacobson I. New direct-acting antiviral agents for the treatment of hepatitis C virus infection and perspectives. Gut 2012;61 Suppl 1:i36-46.
David S, Sachithanandham J, Jerobin J, Parasuram S, Kannangai R. Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South). Indian J Med Microbiol 2012;30:403-6.
] [Full text]
Marins EG, Bodinaidu K, Lin M, Deforest A. Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV test v2.0 for HCV viral load monitoring using dried blood spot specimens. J Virol Methods 2017;247:77-80.
Kamal SM. Acute hepatitis C: A systematic review. Am J Gastroenterol 2008;103:1283-97.
Dokubo EK, Evans J, Winkelman V, Cyrus S, Tobler LH, Asher A, et al.
Comparison of hepatitis C virus RNA and antibody detection in dried blood spots and plasma specimens. J Clin Virol 2014;59:223-7.
Abe K, Konomi N. Hepatitis C virus RNA in dried serum spotted onto filter paper is stable at room temperature. J Clin Microbiol 1998;36:3070-2.
Solmone M, Girardi E, Costa F, Pucillo L, Ippolito G, Capobianchi MR. Simple and reliable method for detection and genotyping of hepatitis C virus RNA in dried blood spots stored at room temperature. J Clin Microbiol 2002;40:3512-4.
Tuaillon 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.
Bennett S, Gunson RN, McAllister GE, Hutchinson SJ, Goldberg DJ, Cameron SO, et al.
Detection of hepatitis C virus RNA in dried blood spots. J Clin Virol 2012;54:106-9.
Brandão CP, Marques BL, Marques VA, Villela-Nogueira CA, Do Ó KM, de Paula MT, et al.
Simultaneous detection of hepatitis C virus antigen and antibodies in dried blood spots. J Clin Virol 2013;57:98-102.
Cloherty GA, Young TP, Lucic D, Steinhart C, Letendre S. Dried blood spots (DBS) for hepatitis C (HCV) molecular diagnostic testing. J Hepat Res 2014;1:1017.
Soulier A, Poiteau L, Rosa I, Hézode C, Roudot-Thoraval F, Pawlotsky JM, et al.
Dried blood spots: A Tool to ensure broad access to hepatitis C screening, diagnosis, and treatment monitoring. J Infect Dis 2016;213:1087-95.
Neesgaard B, Ruhwald M, Krarup HB, Weis N. Determination of anti-HCV and quantification of HCV-RNA and IP-10 from dried blood spots sent by regular mail. PLoS One 2018;13:e0201629.
Mahajan S, Choudhary MC, Kumar G, Gupta E. Evaluation of dried blood spot as an alternative sample collection method for hepatitis C virus RNA quantitation and genotyping using a commercial system. Virusdisease 2018;29:141-6.
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;36:230-5.
] [Full text]
Marques BL, do Espírito-Santo MP, Marques VA, Miguel JC, da Silva EF, Villela-Nogueira CA, et al.
Evaluation of dried blood spot samples for hepatitis C virus detection and quantification. J Clin Virol 2016;82:139-44.
Mössner BK, Staugaard B, Jensen J, Lillevang ST, Christensen PB, Holm DK. Dried blood spots, valid screening for viral hepatitis and human immunodeficiency virus in real-life. World J Gastroenterol 2016;22:7604-12.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]