Alvarez et al., J Virol Antivir Res 2014, 3:2 http://dx.doi.org/10.4172/2324-8955.1000123
Journal of Virology & Antiviral Research
Research Article
HIV-1 RNA Quantification from Dried Blood Spots and Plasma Using the Siemens VERSANT® HIV-1 RNA 1.0 Assay (kPCR) Patricia Alvarez1, Carmen Rodríguez2, Leticia Martín1, Jorge del Romero2 and África Holguín1*
Abstract Objective: Dried blood specimens (DBS) use simplifies the sample collection for viral load (VL) testing in some settings. We compared the VL quantification using DBS and the plasma using the Siemens VERSANT® HIV-1 RNA 1.0 (kPCR) Assay. Methods: Paired DBS/plasma samples were prepared from 62 HIV-1 infected patients harboring different HIV-1 subtypes and recombinants and HIV-1 RNA was quantified by kPCR in 62 paired specimens. DBS and plasma VL results were compared. Viraemia values in DBS were corrected for plasma volume differences assuming the hematocrit percentage in each patient. Results: The results showed a good correlation between corrected VL values in DBS and the results obtained in plasma. The intraclass correlation coefficient was 82.3%. The detection rate in DBS was 83.9%, while the sensitivity of the kPCR for DBS was 92.9%. Overall VL in DBS was, on average, 0.8 log (SD = 0.58) lower than in plasma. We observed overestimated VL in DBS specimens with less than 1,000 copies/ml in plasma. Conclusion: DBS can be used for HIV-1 quantification using kPCR and can be useful for the early therapeutic failure detection in treated subjects. However, VL in DBS can be overestimated in specimens with low VL in plasma, maybe due to a higher effect of proviral DNA in quantification. More studies including more HIV-1 variants are needed to define the kPCR performance.
Keywords HIV-1; Viral load; Dried blood spot testing; Plasma; Kinetic polymerase chain reaction; Subtypes; Recombinants
Abbreviations DBS: Dried Blood Specimens; VL: Viral Load; SD: Standard Deviation; HIV-1: Human Immunodeficiency Virus Type 1; ART: Antiretroviral Therapy; PCR: Polymerase Chain Reaction; CRF: Circulating Recombinant Form; DNA: Deoxyribonucleic Acid; RNA: Ribonucleic Acid; μl: Microliter; ml: Milliliter; ICC: Intra-class Correlation Coefficient; log: Logarithm; CI: Confidence Interval; WHO: World Health Organization.
*Corresponding author: África Holguín, HIV-1 Molecular Epidemiology Laboratory, Microbiology Department, Hospital Universitario Ramón y CajalIRYCIS, Carretera de Colmenar Km 9,100, 28034 Madrid, Spain, Fax: +34 91 3368153; E-mail:
[email protected] Received: May 13, 2014 Accepted: June 04, 2014 Published: June 06, 2014
International Publisher of Science, Technology and Medicine
a SciTechnol journal
Introduction Human immunodeficiency virus type 1 (HIV-1) viral load (VL) testing is useful for monitoring the efficacy of antiretroviral therapy (ART), detecting early therapeutic failure events and performing an early diagnosis (4-6 weeks of life) of the HIV infection in infants [1,2]. Dried blood specimens (DBS) represent an alternative sample type to liquid plasma for performing HIV-1 monitoring techniques since they are easy to prepare, less biohazardous, do not require a cold chain and can be shipped by standard mail at room temperature to a reference laboratory if required. DBS collection is especially useful in settings with limited infrastructures or when low blood volume is available, as in infants [3-5]. The VERSANT HIV-1 RNA 1.0 Assay (kPCR) (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) quantifies HIV-1 RNA by real time polymerase chain reaction (PCR). It detects a highly conserved region within the viral Integrase [6], with a detection limit of 37 HIV-1-RNA copies/ml (1.57 log/ml) in plasma and 866 copies/ ml in DBS [7,8], which is the lowest VL value to generate positive results using one spot of dried blood. However, the effect of HIV1 subtype in the performance of kPCR has not been extensively evaluated. This assay has quantified some HIV-1 variants in plasma specimens [6,9], but not in DBS [7,8,10]. Very few studies reporting VL obtained from DBS consider the hematocrit percentage for correcting viraemia values [11,12], and none of them was performed using kPCR assay. Due to genetic differences, HIV-1 Group M has been classified in nine subtypes (A,B,C,D,F,G,H,J,K), in at least 65 circulating recombinant forms (CRF) and multiple unique recombinants [13,14]. HIV-1 non-subtype B viruses cause nearly 90% of the 35 million infections worldwide. The high HIV-1 genetic heterogeneity and the emergence of new recombinant viruses complicate the performance of molecular techniques detecting viral RNA and/or proviral DNA, including VL assays [15-18]. In fact, HIV-1 viral variability can lead to VL underestimation or to no HIV-1 RNA detection using different methodologies [6,9,15-17,19], as reported in other RNA viruses [20]. The main objective of this study was to evaluate the agreement between HIV-1 viraemia quantification using paired plasma/DBS specimens by the kPCR assay in infected subjects carrying different HIV-1 variants.
Materials and Methods Both plasma and DBS specimens were collected from 62 wellcharacterized HIV-infected ART naive adults attending the Centro Sanitario Sandoval (Madrid, Spain) from May 2011 to March 2012. Patients came from 14 different countries, mainly Spain (58.1%) and Latin-America (33.9%). All individuals signed the informed consent and the local ethics committee approved the study protocol. Viral sequences encoding the complete HIV-1 protease gene (codons 1-99) and partial reverse transcriptase gene (codons 1-335) perfomed with ViroSeq HIV-1 Genotyping System (Abbott Molecular, USA) were available in 56 subjects. They were used to characterize the HIV-1 variant using three available online subtyping tools: Stanford HIVdb 6.0.5 (http://hivdb.stanford.edu/pages/algs/sierra sequence.html, Stanford University, Palo Alto, CA), Geno2Pheno (http://www.geno2pheno.
All articles published in Journal of Virology & Antiviral Research are the property of SciTechnol, and is protected by copyright laws. Copyright © 2014, SciTechnol, All Rights Reserved.
Citation: Alvarez P, Rodríguez C, Martín L, Romero J, Holguín A (2014) HIV-1 RNA Quantification from Dried Blood Spots and Plasma Using the Siemens VERSANT® HIV-1 RNA 1.0 Assay (kPCR). J Virol Antivir Res 3:2.
doi:http://dx.doi.org/10.4172/2324-8955.1000123 org, Max Planck Institute for Informatics, Saarbrücken, Germany) and Comet (http://comet.retrovirology.lu/, Centre de Recherche Public de la Santé, Luxembourg). HIV-1 non-B variants infected 11 (19.6%) of 56 subjects: 5 from Spain, 5 from Latin America and 1 from Angola. DBS were prepared by adding 70 μl of venous blood, collected by venipuncture in 5 ml EDTA-anticoagulated tubes to one spot of filter paper (Whatman no.903). Plasma was collected from the remaining blood. DBS were dried overnight at room temperature, placed in individual zip-lock bags containing a silica desiccant and stored at -80ºC until VL testing. The spot was incubated for 30 minutes at room temperature with gentle rotation in 2 ml of DBS-specific lysis buffer including guanidine thiocyanate (Siemens Healthcare Diagnostics, not available for sale). The tubes were centrifuged for 2 minutes at room temperature. Then, 1.1 ml of clean supernatant was processed with the VERSANT® Sample Preparation Module. The same kPCR software and settings were used for both DBS and plasma samples with no modification. Viraemia values in DBS were corrected for plasma volume differences, the hematocrit percentage being assumed in each patient. The hematocrit value was obtained using SYSMEX XT-1800i automated analyzer. The dried blood has both cellular and plasma fractions. The hematocrit, or packed cell volume, is the volume percentage of red blood cells in blood. Thus, the plasma volume percentage in 70 µl of dried blood in each subject can be assumed to be 100 minus the hematocrit percentage value. Hence, to compare VL in plasma and in DBS, the HIV-1-RNA copies provided by kPCR using one dot of DBS should be expressed in HIV-1-RNA copies per ml of plasma in the 70 µl of dried blood. Thus, the corrected viral load in DBS by plasma volume in the dot considering the hematocrit value in each patient was calculated as follows: corrected number of RNA copies per milliliter of plasma in the DBS spot = obtained number of RNA copies in the kPCR using one dot x 1,000/[70 – (70 x hematocrit value/100)]. Before performing statistical analyses, the VL results obtained were converted to log10 values. HIV-RNA values below the detection limit (
