|Year : 2012 | Volume
| Issue : 4 | Page : 403-406
Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South)
S David, J Sachithanandham, J Jerobin, S Parasuram, R Kannangai
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu-632004, India
|Date of Submission||01-May-2012|
|Date of Acceptance||26-Jun-2012|
|Date of Web Publication||24-Nov-2012|
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu-632004
Source of Support: Authors like to thank Christian Medical College Fluid Research Fund for the partial financial support for this study, Conflict of Interest: None
Purpose: The use of dried blood spots (DBS) for HIV-1 viral load determination could greatly enhance the management of HIV infected individuals in resource-limited countries. Objective: To compare the HIV-1 viral load values obtained between parallel collected plasma and DBS. Materials and Methods: DBS and plasma samples were collected from 62 HIV-1 infected individuals and were used for determination of HIV-1 RNA concentrations using the Abbot real-time HIV-1 PCR. Result: Mean of the log difference of viral load values between plasma and DBS was -0.41 log. DBS viral load values significantly correlated with plasma viral load (r = 0.9818, P < 0.0001). Conclusion: These results suggest that DBS samples can be used as an alternative to plasma for the estimation of HIV-1 viral load if samples are appropriately stored.
Keywords: Dried blood spot, HIV-1 RNA, India, real-time PCR
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2012 [cited 2020 Aug 5];30:403-6. Available from: http://www.ijmm.org/text.asp?2012/30/4/403/103759
| ~ Introduction|| |
Globally, approximately 33.4 million persons are living with human immunodeficiency virus (HIV) disease.  The national adult HIV prevalence for India was 0.36% (range 0.29-0.46%) and the estimated number of people living with HIV was 2.47 million (range 2.0-3.1million) in 2006.  Despite the downward trend of HIV infection in the country, India continues to face appalling challenge to provide prevention, treatment, and care to those in need. 
PCR-based nucleic acid amplification and quantification of HIV-1 is the gold standard for evaluating antiretroviral therapy (ART) efficacy of patients on ART and also for early HIV-1diagnosis in infants. , The use of PCR-based nucleic acid amplification is constrained in developing countries, due to the lack of sufficient facilities and equipment, and the difficulty involved with the cryopreservation of plasma during transportation and storage at the tropical temperature. Practical and dependable methods to obtain, store, and transport blood samples are necessary to develop cost effective early diagnostic assays in limited-resource settings.  In resource-limited countries the number of facilities to measure HIV-1 viral load is inadequate. Whole blood can easily be spotted onto filter paper from finger or heel stick punctures when bleeding neonates, offers technical and economic advantage over conventional vein puncture methods. It simplifies sample collection, thus avoiding the use of syringes and vacutainer tubes and reducing the risks associated with it and the need for centrifugation.  HIV-1, HIV-2, Hepatitis C virus that are known to be present in serum, lose infectivity due to the disruption of envelope on drying. Dried blood spots (DBS) can be readily shipped in sealed envelopes to reference centers, whereas, sera need to be transported in break-proof containers. Transport of frozen sera may require the use of dry ice or liquid nitrogen, maintaining cold chain, requiring further specialized handling and adding considerable weight to the item. Sterilities are available at reduced price than sterile disposable needles. 
There are several reports on the use of DBS as an alternative method to plasma for HIV viral load estimation.  DBS have been successfully used in confirmation of HIV serological diagnosis, the ultrasensitive p24 antigen assay, measurement of CD4 counts, to detect drug resistance mutations and even analyses of antiretroviral drug levels. ,,,,, However, there is insufficient data currently available for the Indian subcontinent on the utility of DBS as a sample for HIV-1 viral load assay.
This necessitated the need to undertake a pilot study to see the correlation between viral load on plasma samples and DBS and to see whether DBS can be used as an alternative sample to plasma for viral load estimation.
| ~ Materials and Methods|| |
After Institutional Review Board clearance, 62 blood samples were collected, during November 2009 and May 2012, from HIV-infected individuals, coming to the Clinical Virology department of a tertiary care center in South India. Blood was collected, from patients coming for routine CD4 and HIV-1 viral load testing, who are treatment naïve. Samples were also collected from patients who were on treatment and or were referred to us for genotypic HIV-1 drug resistance testing.
Around 4 ml of whole blood samples was collected by vacutainer system into K2 EDTA tubes, of which 50 μl each was applied on Whatman 903 filter paper cards (Whatman, Sanford, CA, USA) on five spots. Blood spots were dried keeping at room temperature for 6-8 h. After drying it was stored with desiccants pouches in -20°C for a period of 1-6 months.
In parallel plasma was also separated from each sample and stored in multiple aliquots at -70°C for a period of 1-6 months.
Sample Preparation and RNA Extraction
The total RNA was extracted from plasma and DBS specimen by Abbott m2000sp automated system (Abbot Diagnostics Gmbh, Wiesbaden, Germany). For the RNA extraction, two half-inch disks (6 mm in diameter) entirely covered with the sample from the DBS card was cut using a puncher and it was transferred by clean forceps to a 50 ml sterile screw cap conical tube containing 1.7 ml of lysis buffer provided by the manufacturer. Then the tubes were incubated at room temperature (15-30°C) for 2 h, with intermittent mixing. The tubes were spun at 5000 rpm for 1 min and then the whole viral lysate were transferred to an m2000sp reaction vessel using micropipette. The lysates was processed according to the standard HIV-1 RNA 1 ml extraction protocol provided by the manufacturer. Appropriate dilution factor was taken into consideration while calculating the copy number/ml. The lower limit of detection of this assay was 40 copies/ml.
Plasma samples were vortexed and centrifuged at 5000 rpm for 1 min at room temperature. A minimum of 350 μl of plasma was transferred to an m2000sp reaction vessel and processed using the 0.2 ml protocol as per the manufacturer's instruction. In the 0.2 ml Abbott protocol, the lower limit of detection was 150 copies/ml. The ten samples from treatment experienced individuals were tested with 1.0 ml protocol and the detection limit was 40 copies/ml. Following extraction all the processed samples were then transferred to the Abbott m2000sp automated system and processed as per the manufacturer's instructions.
This difference in the lower detection limit was due to the difference in the protocol used in the Abbott system for DBS and plasma samples. We did not have enough volume of plasma sample (1 ml) to use the protocol with the lower detection limit of 40 copies/ml and hence used the 0.2 ml protocol.
| ~ Haematocrit Determinations and Calculation of Hematocrit-Corrected DBS Viral Load|| |
Since the DBS has cellular and plasma fractions, a correction factor was used for calculating the hematocrit. The hematocrit values for all the individuals were not available and hence the mean of the hematocrit value was calculated with the available data (n = 22) and the hematocrit value was 34. Since in DBS, the actual input volume is 100, the component of blood, that is, plasma, can be assumed to be the hematocrit subtracted from 100, that is, 100-34 = 66. The correction factor used was 1.5, that is, 100/66.
Statistical analysis was done using the Bland and Altman method and also by Pearson correlation analysis using Stata software version 10.
| ~ Results|| |
Overall, HIV-1 RNA levels obtained from DBS were always lower than in plasma. Viral load values between DBS and plasma differed by less than 0.5 log values in 52% (27/52) of the samples and between 0.5 and 1 log units in 44% (23/52) of the samples. The difference in log value of >1 occurred in two (3.8%) of the samples. In addition, in all the 10 samples, the plasma HIV-1 RNA was undetectable the DBS also gave concordant results. The maximum number of viral load values for DBS and plasma were obtained in the range of 10,000-50,000 and >50,000 copies/ml. Mean of the log difference of viral load values between plasma and DBS is - 0.41. When a viral load of 50,000 copies/ml was considered as an arbitrary cut-off for comparison, the observed agreement between the plasma viral load and DBS viral load was 88% with kappa coefficient of 0.87 (P < 0.0001).
Agreement between the two methods was also analyzed by the Bland and Altman method, in which log of the average of plasma viral load results and DBS are plotted against each other (concordance is good when the differences are within the limits of 1.96 standard deviations of the mean). The Bland-Altman analysis showed that 98.3% of the results were within two standard deviation (-1.96 and + 1.96) as shown in [Figure 1]. Log-transformed viral load values measured in the DBS samples and in the paired liquid plasma samples were compared by Pearson's correlation test and there was a significant correlation (r = 0.9818, P = < 0.0001). This data is shown in [Figure 2].
|Figure 1: Bland– Altman analysis of agreement between plasma and DBS. The horizontal lines represent the mean difference and ± 1.96 standard deviations|
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|Figure 2: Correlation between human immunodeficiency virus type 1 (HIV-1) RNA levels measured with the Abbot real-time assay in dried blood spot (DBS;Y-axis) and liquid plasma (X-axis) samples|
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| ~ Discussion|| |
Evaluating the reliability of DBS for the detection of HIV viral load is of high priority in resource-limited settings in tropical countries. Various studies conducted in different parts of the world, have found good correlation between HIV-1 viral loads detected from DBS and paired plasma samples. ,, In a study conducted in Dakar, HIV-1 RNA concentrations in plasma were compared with concentrations in DBS after 8 and 15 days. The study showed a strong concordance [r = 0.98 (8 days) and 0.94 = (15 days)] in RNA levels between plasma and DBS. 
Several other studies reported from Asia, Europe, and Africa had shown a significant correlation between the HIV-1 viral RNA level estimated with plasma and DBS with an 'r' value varying from 0.75 to 0.88. ,, In comparison to the above studies our study also showed a very good correlation with an 'r' value of 0.9818 (P < 0.0001).
Bland and Altman comparison of DBS and plasma viral load data from an European study showed 99.22% of the tested samples were within the 1.96 standard deviation (-0.30 and -0.94).  A study done in Africa had also shown that 94% and 97% of results were within two standard deviation on day 8 and 15, respectively, when comparing plasma and DBS viral load results.  Similar to the above mentioned studies, Bland-Altman analysis of our data showed 98.3% of the results within 1.96 standard deviation (0.27 and -1.09). When plasma and DBS viral load of more than 50,000 copies/ml was considered as an arbitrary cut-off, observed agreement between the two techniques in our study was 88% with kappa coefficient of 0.87 (P < 0.0001).
In a study conducted in Thailand, when the DBS viral load was corrected for volume only, the r-value was 0.949 and the mean of the log difference of viral load values between plasma and DBS was 0.428. When it was corrected for volume and heamatocrit, the r value was 0.949, and the mean difference was 0.283.  In our study, the heamatocrit for all the samples were not done, hence the mean of the heamatocrit of the available 22 samples was obtained. When corrected for heamatocrit and volume, a high correlation with plasma viral load was obtained with 'r' value being 0.98 and the mean difference of viral load being 0.41 log. Wherever available, the individual hematocrit value was used to correct the available DBS viral load value. On comparing the DBS viral load value obtained with the mean hematocrit value (n = 22) and the individual heamatocrit value, the difference in the viral load was not significant (P value = 0.08)
An earlier study, done in Italy, has found high correlation between plasma and DBS, using Abbot real time HIV-1 assay.  Recently there was a study reported from south India using the same Abbott system, which showed a mean log difference of 0.14 between plasma and DBS samples and a significant correlation with an 'r' value of 0.86.  Our study is very similar to the two above-mentioned studies.
In our study, viral load values between DBS and plasma differed by less than 0.5 log values in 52% of the samples and between 0.5 and 1 log units in 44% of the samples. In the Marconi et al. study, 78.5% of cases differed by < 0.5 log and 99.4% of cases by 1 log in paired DBS and plasma samples.  In the study by Andreotti et al., 78.4% of samples differed by less than 0.5 log unit and by less than 1 log unit in 100% of samples.  The recently reported study from south India showed that 86% of the their results were within 0.5 log copies/ml compared with plasma samples and 95% of the results were within 1.96 standard deviation in Bland-Altman analysis.  In our study majority (73%) of the >0.5 log difference were seen when the viral load level was more than 5.0 log copies/ml.
In our study the sample with lowest viral load tested was 3.47 log copies/ml and for the same sample the DBS viral load estimated was 4.17 log copies/ml. This shows the DBS can accurately amplify a viral load level of >3000 (3.47 log) RNA copies. As per the 2009 WHO guideline, the threshhold for virological failure or the recommendation for ART regimen change is persistent viral load of >5000 (3.7 log) copies/ml in resource poor settings. , In our work, unlike other reports, we did not find any false positive or false negative results with DBS. Hence, DBS can be used as an alternative to plasma samples to monitor the HIV-1 RNA level in an infected individual in resource poor settings.
In conclusion, our pilot study, showed a good correlation between plasma and DBS viral load estimation. More studies need to be done with much larger sample size and the stability of DBS samples at various temperatures in a tropical country like ours also should be investigated. DBS is a very reliable method to store and transport blood samples for viral load assays in tropical conditions and is very user friendly and cost effective in resource poor settings.
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[Figure 1], [Figure 2]