HIV type-1 (HIV-1) drug resistance testing is rarely available in resource-limited settings because of high costs and stringent requirements for storage and transport of plasma. Dried blood spots (DBS) can be a convenient alternative to plasma, but the use of DBS needs validation under field conditions. We assessed the performance of DBS in genotypic resistance testing of patients who failed first-line antiretroviral therapy (ART) in rural Tanzania.
Methods
A total of 36 ART-experienced patients with viral loads >1,000 copies/ml (median 15,180 copies/ml [range 1,350–3,683,000]) and with various HIV-1 subtypes were selected for resistance testing. DBS were stored with desiccant at ambient temperature for a median of 29 days (range 8–89). Samples were amplified using an in-house reverse transcriptase-nested PCR method and sequenced using the ViroSeq™ assay (Abbott Molecular, Des Plaines, IL, USA). DBS-derived genotypes were compared with genotypes from plasma.
Results
Overall, 34 of 36 (94%) DBS specimens were successfully genotyped. In the protease region, of 142 polymorphisms found in plasma, 132 (93%) were also detected in DBS. In the reverse transcriptase region, of 57 clinically relevant mutations present in plasma, 51 (89%) were also detected in DBS. A total of 30 of 34 (88%) patients had identical resistance profiles to antiretroviral drugs in plasma and DBS.
Conclusions
Genotyping was successful in the vast majority of DBS specimens stored at ambient temperature for up to 3 months, and there was high concordance between mutations found in DBS and plasma. Our study suggests that DBS can be a feasible and reliable tool to monitor HIV-1 drug resistance in patients on ART in resource-limited settings.
References
1.
World Health Organization.Towards universal access. Scaling up priority HIV/AIDS interventions in the health sector. Progress report 2009. Geneva: World Health Organization2009.
2.
Bussmann H, Wester CW, Ndwapi N, et al.Five-year outcomes of initial patients treated in Botswana's National Antiretroviral Treatment Program.AIDS2008; 22: 2303–2311.
3.
LawnS.D., MyerL., OrrellC., BekkerL.G., WoodR.Early mortality among adults accessing a community-based antiretroviral service in South Africa: implications for programme design.AIDS2005; 19: 2141–2148.
4.
NashD., KatyalM., BrinkhofM.W.Long-term immunologic response to antiretroviral therapy in low-income countries: a collaborative analysis of prospective studies.AIDS2008; 22: 2291–2302.
5.
HammerS.M., EronJ.J.Jr., ReissP.Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society–USA panel.JAMA2008; 300: 555–570.
6.
SungkanuparphS., ManosuthiW., KiertiburanakulS., PiyavongB., ChumpathatN., ChantratitaW.Options for a second-line antiretroviral regimen for HIV type 1-infected patients whose initial regimen of a fixed-dose combination of stavudine, lamivudine, and nevirapine fails.Clin Infect Dis2007; 44: 447–452.
7.
WallisC.L., MellorsJ.W., VenterW.D., SanneI., StevensW.Varied patterns of HIV-1 drug resistance on failing first-line antiretroviral therapy in South Africa.J Acquir Immune Defic Syndr2010; 53: 480–484.
8.
MeiJ.V., AlexanderJ.R., AdamB.W., HannonW.H.Use of filter paper for the collection and analysis of human whole blood specimens.J Nutr2001; 131: 1631S–1636S.
9.
BrambillaD., JenningsC., AldrovandiG.Multicenter evaluation of use of dried blood and plasma spot specimens in quantitative assays for human immunodeficiency virus RNA: measurement, precision, and RNA stability.J Clin Microbiol2003; 41: 1888–1893.
10.
LofgrenS.M., MorrisseyA.B., ChevallierC.C.Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities.AIDS2009; 23: 2459–2466.
11.
MasciotraS., GarridoC., YoungpairojA.S.High concordance between HIV-1 drug resistance genotypes generated from plasma and dried blood spots in antiretroviral-experienced patients.AIDS2007; 21: 2503–2511.
12.
BertagnolioS., Soto-RamirezL., PilonR.HIV-1 drug resistance surveillance using dried whole blood spots.Antivir Ther2007; 12: 107–113.
13.
McNultyA., JenningsC., BennettD.Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing.J Clin Microbiol2007; 45: 517–521.
14.
HallackR., DohertyL.E., WethersJ.A., ParkerM.M.Evaluation of dried blood spot specimens for HIV-1 drug-resistance testing using the Trugene HIV-1 Genotyping Assay.J Clin Virol2008; 41: 283–287.
15.
YoungpairojA.S., MasciotraS., GarridoC., ZahoneroN., de MendozaC., Garcia-LermaJ.G.HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C.J Antimicrob Chemother2008; 61: 1217–1220.
16.
Garcia-LermaJ.G., McNultyA., JenningsC., HuangD., HeneineW., BremerJ. W.. Rapid decline in the efficiency of HIV drug resistance genotyping from dried blood spots (DBS) and dried plasma spots (DPS) stored at 37 degrees C and high humidity.J Antimicrob Chemother2009; 64: 33–36.
17.
HearpsA.C., RyanC.E., MorrisL.M., PlateM.M., GreengrassV., CroweS.M.Stability of dried blood spots for HIV-1 drug resistance analysis.Curr HIV Res2010; 8: 134–140.
18.
World Health Organization.Scaling up antiretroviral therapy in resource-limited settings. Guidelines for a public health approach. Geneva: World Health Organization2002.
19.
World Health Organization.Scaling up antiretroviral therapy in resource-limited settings: treatment guidelines for a public health approach. 2003 revision. Geneva: World Health Organization2004.
20.
World Health Organization.Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach. 2006 revision. Geneva: World Health Organization2006.
21.
JohannessenA., NamanE., KivuyoS.L.Virological efficacy and emergence of drug resistance in adults on antiretroviral treatment in rural Tanzania.BMC Infect Dis2009; 9: 108.
22.
NyombiB.M., KristiansenK.I., BjuneG., MullerF., Holm-HansenC.Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania.AIDS Res Hum Retroviruses2008; 24: 761–769.
23.
JohnsonV.A., Brun-VezinetF., ClotetB.Update of the drug resistance mutations in HIV-1: December 2009.Top HIV Med2009; 17: 138–145.
BoomR., SolC.J., SalimansM.M., JansenC.L., Wertheim-van DillenP.M., van der NoordaaJ.Rapid and simple method for purification of nucleic acids.J Clin Microbiol1990; 28: 495–503.
26.
ZiemniakC., George-AgwuA., MossW.J., RayS.C., PersaudD.A sensitive genotyping assay for detection of drug resistance mutations in reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried blood spots or frozen RNA extracts.J Virol Methods2006; 136: 238–247.
27.
BertagnolioS., DerdelinckxI., ParkerM.World Health Organization/HIVResNet Drug Resistance Laboratory Strategy.Antivir Ther2008; 13 Suppl 2: 49–57.
28.
BiX., GatanagaH., IdaS.Emergence of protease inhibitor resistance-associated mutations in plasma HIV-1 precedes that in proviruses of peripheral blood mononuclear cells by more than a year.J Acquir Immune Defic Syndr2003; 34: 1–6.
29.
VenturiG., RomanoL., CarliT.Divergent distribution of HIV-1 drug-resistant variants on and off antiretroviral therapy.Antivir Ther2002; 7: 245–250.
30.
CalmyA., FordN., HirschelB.HIV viral load monitoring in resource-limited regions: optional or necessary?Clin Infect Dis2007; 44: 128–134.
31.
FiscusS.A., BrambillaD., GrossoL., SchockJ., CroninM.Quantitation of human immunodeficiency virus type 1 RNA in plasma by using blood dried on filter paper.J Clin Microbiol1998; 36: 258–260.
32.
GarridoC., ZahoneroN., CorralA., ArredondoM., SorianoV., de MendozaC.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 Microbiol2009; 47: 1031–1036.
33.
JohannessenA., GarridoC., ZahoneroN.Dried blood spots perform well in viral load monitoring of patients who receive antiretroviral treatment in rural Tanzania.Clin Infect Dis2009; 49: 976–981.
34.
MbidaA.D., SossoS., FloriP.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 Syndr2009; 52: 9–16.
