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Year : 2012  |  Volume : 136  |  Issue : 6  |  Page : 903-905

Dried blood spot testing: filling the gap between antiretroviral treatment & monitoring in India

Department of Medical Biotechnologies University of Siena Viale Bracci 11-53100 Siena, Italy

Date of Web Publication4-Feb-2013

Correspondence Address:
M Zazzi
Department of Medical Biotechnologies University of Siena Viale Bracci 11-53100 Siena
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Source of Support: None, Conflict of Interest: None

PMID: 23391786

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How to cite this article:
Zazzi M. Dried blood spot testing: filling the gap between antiretroviral treatment & monitoring in India. Indian J Med Res 2012;136:903-5

How to cite this URL:
Zazzi M. Dried blood spot testing: filling the gap between antiretroviral treatment & monitoring in India. Indian J Med Res [serial online] 2012 [cited 2021 Apr 19];136:903-5. Available from:

Antiretroviral coverage in low-middle income countries has significantly been expanded in recent years, substantiated in the ambitious United Nations commitment to complete universal access to HIV care by 2015 [1] . While decreased cost of drugs, availability of generic drugs, expanded foreign assistance and improved local health policies are expected to facilitate this goal, a key paradigm of the antiretroviral treatment (ART) success achieved in western countries is the integration between therapy and virological monitoring [2] . Specifically, quantification of HIV RNA in plasma (viraemia) has been long recognized as the key surrogate parameter of the course of HIV infection. The definition of virological failure or success of antiretroviral treatment is indeed based on plasma HIV RNA itself. Viraemia and CD4 cell counts are the reference parameters, together with genotypic drug resistance testing [3] , to obtain the maximum benefit from access to therapy.

Measurement of HIV RNA requires the availability of a robust molecular system certified for in vitro diagnostic use and the possibility to store and send plasma samples in a well-controlled cold chain system. One alternative to processing and shipment of plasma is to spot and dry blood on filter paper and have HIV RNA retrieved and measured later from this dried blood spot (DBS) matrix. The advantage of the system is that nucleic acids are relatively stable in DBS and thus the requirement for a cold chain during storage and transportation is not necessary. This in turn facilitates access to HIV RNA testing in resource-limited settings, particularly rural areas where local laboratory facilities are scarce or absent and the reference laboratory is difficult to reach. Measurement of HIV RNA in DBS was first described in 1997 [4] and has been increasingly documented as a reliable methodology in recent years through simulated experiments in western countries or field testing in low-income countries, particularly in Africa [5],[6] . Importantly, the latest studies have all adapted certified state-of-the-art HIV RNA assays [7],[8],[9],[10] rather than developing in house methods. The latter, although attractive in terms of cost reduction, may add the need for an extra optimization effort and potentially lead to lower accuracy with respect to certified commercial systems.

The paper by Neogi et al[11] in this issue follows this line showing feasibility and accuracy of DBS HIV RNA quantification via the Abbott m2000rt assay in the Indian settings. The results are very similar to those reported by the same method few months ago by another Indian group [12] . Both studies used manual extraction, instead of the integrated m2000sp automated platform usually coupled with this system, yet the correlation with the reference liquid plasma HIV RNA values was high. Neogi et al[11] showed that the difference in viraemia between the paired samples was within 0.5 log in 80 per cent of the cases and >1.0 log in no case, similar to what also the other Indian group reported earlier [12] . Among current HIV RNA assays, the Abbott system is the one which has been used on DBS more frequently [7],[11],[12],[13],[14],[15] .

In general, most of the studies comparing DBS vs. liquid plasma for HIV RNA quantification have so far yielded satisfactory results, supporting the use of DBS when plasma cannot be properly stored and shipped. The limitations of the DBS approach have also been reviewed [16] . First, the small volume of blood spotted (usually 50 microliters per spot) and the low efficiency of RNA extraction from multiple spots significantly limit the concentration of the RNA extract with respect to standard processing of liquid plasma. This results in lower sensitivity of DBS testing, with detection of HIV RNA in about 50 per cent of the cases where plasma HIV RNA is around 3 log. Second, correlation with plasma HIV RNA values, and thus accuracy, decreases with lower viraemia levels. This is partly due to the same reason (small blood volume analyzed) and partly complicated by the inability to distinguish between HIV RNA and DNA which can falsely increase the test result [12] . Third, although nucleic acids in DBS are relatively stable, detailed studies [17],[18] have reported variable HIV RNA degradation with prolonged storage at room temperature, particularly in high humidity conditions that can be frequently found in many resource-limited settings.

The third issue appears to be easily addressed, assuming that even remote rural regions can store DBS in a refrigerator as long as needed. In addition, DBS HIV RNA testing in clinical practice must be performed as timely as possible to be clinically useful. Thus, prolonged storage would probably not occur in this setting, limiting the impact even of storage at high temperature. On the other hand, the issues related to the low amount of blood analyzed and the unpredictable level of contaminating HIV DNA in the DBS do not have an easy technical solution. HIV DNA could be removed by DNase digestion but this requires an extra step currently not included in any certified HIV RNA quantification system (because it is not needed when working with plasma) and would considerably complicate DBS handling. In theory, the amount of plasma, and hence the assay sensitivity, could be increased by processing multiple DBS. However, collection and excision of many spots is highly impractical and, when we first adapted the Abbott assay to DBS [7] , we found no increase in RNA concentration using more than four spots because of the annoyances with a large amount of paper requiring large volumes of buffer (personal data). Limited sensitivity and possible inaccuracies due to contaminating HIV DNA at low HIV RNA levels result in difficulty to detect early virological failure of antiretroviral treatment, i. e. low-level HIV RNA rebound. At this time, it is difficult to anticipate what, if any, can be the consequences of a potentially delayed diagnosis of virological treatment failure in the resource-limited setting. Although remaining on a failing regimen for a prolonged period carries an inherent risk of drug resistance and cross-resistance development [19] , the sensitivity of DBS HIV RNA testing should be enough to provide an effective guide to treatment change when needed.

In summary, the potential drawbacks associated with the use of DBS for HIV RNA quantification appear to be largely outweighed by the benefits derived from increased access to virological monitoring of antiretroviral treatment in resource-limited settings. To warrant adequate accuracy, certified HIV RNA quantification systems should be probably preferred over in house technology, unless exhaustive optimization and comparative analyses with a reference system have been successfully completed. Also, automated RNA extraction should be preferred over manual systems which, by definition, are much more prone to human error and inconsistencies. Irrespective of the assay used, local authorities should develop the appropriate quality assurance programmes to ensure that the laboratories involved have and maintain a good performance in DBS HIV RNA testing. The use of certified systems and automated extraction procedures clearly requires a larger financial effort with respect to in house assays and manual procedures. However, the expanding use of the reference systems and the possible development of novel certified systems by other companies can reasonably result in much lower costs in a short time. Such cost savings will add to savings derived from avoidance of the cold chain transportation allowed by the DBS format. Thus, quantification of HIV RNA in DBS can be a valid and cost-effective choice in many low-middle income countries aiming at complementing expanded access to antiretroviral treatment with the required virological monitoring.

   References Top

1.United Nations General Assembly. Political Declaration on HIV/AIDS: Intensifying our Efforts to Eliminate HIV/AIDS. July 8, 2011 New York. Available from:, accessed on November 24, 2012.  Back to cited text no. 1
2.Walker AS, Gibb DM. Monitoring of highly active antiretroviral therapy in HIV infection. Curr Opin Infect Dis 2011; 24 : 27-33.  Back to cited text no. 2
3.De Luca A, Prosperi M, Bracciale L. Resistance considerations in sequencing of antiretroviral therapy in low-middle income countries with currently available options. Curr Opin HIV AIDS 2010; 5 : 27-37.  Back to cited text no. 3
4.Cassol S, Gill MJ, Pilon R, Cormier M, Voigt RF, Willoughby B, et al. Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper. J Clin Microbiol 1997; 35 : 2795-801.  Back to cited text no. 4
5.Fiscus 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.  Back to cited text no. 5
6.Mwaba P, Cassol S, Nunn A, Pilon R, Chintu C, Janes M, Zumla A. Whole blood versus plasma spots for measurement of HIV-1 viral load in HIV-infected African patients. Lancet 2003; 362 : 2067-8.  Back to cited text no. 6
7.Marconi A, Balestrieri M, Comastri G, Pulvirenti FR, Gennari W, Tagliazucchi S, et al. Evaluation of the Abbott Real-Time HIV-1 quantitative assay with dried blood spot specimens. Clin Microbiol Infect 2009; 15 : 93-7.  Back to cited text no. 7
8.Pirillo MF, Recordon-Pinson P, Andreotti M, Mancini MG, Amici R, Giuliano M. Quantification of HIV-RNA from dried blood spots using the Siemens VERSANT® HIV-1 RNA (kPCR) assay. J Antimicrob Chemother 2011; 66 : 2823-6.  Back to cited text no. 8
9.van Deursen P, Oosterlaken T, Andre P, Verhoeven A, Bertens L, Trabaud MA, et al. Measuring human immunodeficiency virus type 1 RNA loads in dried blood spot specimens using NucliSENS EasyQ HIV-1 v2.0. J Clin Virol 2010; 47 : 120-5.  Back to cited text no. 9
10.Okonji JA, Basavaraju SV, Mwangi J, Shiraishi RW, Odera M, Ouma K, et al. Comparison of HIV-1 detection in plasma specimens and dried blood spots using the Roche COBAS Ampliscreen HIV-1 test in Kisumu, Kenya. J Virol Methods 2012; 179 : 21-5.  Back to cited text no. 10
11.Neogi U, Gupta S, Rodrigues R, Sahoo PN, Rao SD, Rewari BB, et al. Dried blood spot HIV-1 RNA quantification: A useful tool for viral load monitoring among HIV-infected individuals in India. Indian J Med Res 2012; 136 : 956-62.  Back to cited text no. 11
12.Vidya M, Saravanan S, Rifkin S, Solomon SS, Waldrop G, Mayer KH, et al. Dried blood spots versus plasma for the quantitation of HIV-1 RNA using a real-Time PCR, m2000rt assay. J Virol Methods 2012; 181 : 177-81.   Back to cited text no. 12
13.Garrido C, Zahonero N, Corral A, Arredondo M, Soriano V, de Mendoza C. Correlation between human immunodeficiency virus type 1 (HIV-1) RNA measurements obtained with dried blood spots and those obtained with plasma by use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV load tests. J Clin Microbiol 2009; 47 : 1031-6.  Back to cited text no. 13
14.Mbida AD, Sosso S, Flori P, Saoudin H, Lawrence P, Monny-Lobé M, et al. Measure of viral load by using the Abbott Real-Time HIV-1 assay on dried blood and plasma spot specimens collected in 2 rural dispensaries in Cameroon. J Acquir Immune Defic Syndr 2009; 52 : 9-16.  Back to cited text no. 14
15.Arredondo M, Garrido C, Parkin N, Zahonero N, Bertagnolio S, Soriano V, et al. Comparison of HIV-1 RNA measurements obtained by using plasma and dried blood spots in the automated Abbott real-time viral load assay. J Clin Microbiol 2012; 50 : 569-72.  Back to cited text no. 15
16.Hamers RL, Smit PW, Stevens W, Schuurman R, Rinke de Wit TF. Dried fluid spots for HIV type-1 viral load and resistance genotyping: a systematic review. Antivir Ther 2009; 14 : 619-29.  Back to cited text no. 16
17.Hearps AC, Ryan CE, Morris LM, Plate MM, Greengrass V, Crowe SM. Stability of dried blood spots for HIV-1 drug resistance analysis. Curr HIV Res 2010; 8 : 134-40.  Back to cited text no. 17
18.Monleau M, Butel C, Delaporte E, Boillot F, Peeters M. Effect of storage conditions of dried plasma and blood spots on HIV-1 RNA quantification and PCR amplification for drug resistance genotyping. J Antimicrob Chemother 2010; 65 : 1562-6.   Back to cited text no. 18
19.Hatano H, Hunt P, Weidler J, Coakley E, Hoh R, Liegler T, et al. Rate of viral evolution and risk of losing future drug options in heavily pretreated, HIV-infected patients who continue to receive a stable, partially suppressive treatment regimen. Clin Infect Dis 2006; 43 : 1329-36.  Back to cited text no. 19


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