1. Use of Dried Blood Spots (DBS) Specimens for Measuring HIV-1 Viral Load in Argentina M. Lorena Vázquez Inés Zapiola Ana Gun Silvia Gómez Alejandro Krolewiecki Omar Sued Sara Kaufman Pedro Cahn Centro Médico Huésped Fundación Huésped “Partially funded by Fogarty International Center / NIH” (Grant#D43TW1037)

2. What are DBS (Dried Blood Spots)?: Whole blood Whole blood collected and dried collected and dried on filter paper on filter paper DBS are useful for qualitative detection of HIV antibodies, DNA and RNA. Samples dried on filter paper remain stable for HIV-1 RNA detection. About DBS and HIV, we know… DBS are currently used to determine HIV-1 viral load in some African countries.

3. CDC Recomendations for DBS: Dried blood spot specimens can be shipped or transported by mail or other carriers with no reasonable expectations of occupational exposure to blood or other potentially infectious material. "Universal precautions" are to be followed in collecting and preparing these specimens for shipment. Standard filter paper collection kits should be enclosed and sealed in high quality paper mailers ideally, extra-strong, tear-proof, air-permeable, and water-resistant envelopes. These steps provide reasonable safety against occupational exposure and maintain optimal specimen integrity. (htpp://www.cdc.gov/od/ohs/biosafety/driblood.htm)  DBS can be shipped by regular mail or other carriers  DBS must be enclosed in high quality paper mailers

4. Why Dried blood spots? Laboratory tests are essential for monitoring the efficacy of antivirals and for the optimal management of treatment.  Trained personnel  Centrifugation of samples  Storage of plasma in cryotubes, in freezers  Transport on dry ice  High transportation costs HIV-1 plasmatic viral load Such conditions are often unsuitable for resource-limited countries

5. In Argentina  Shipment of a sample from Orán (Salta) to Buenos Aires by a private courier costs around USD 60 (an additional cost equivalent to the 75% of the value of the viral load) (an additional cost equivalent to the 75% of the value of the viral load)  HIV-1 viral load costs around USD 74

6. Hypothesis :  In resource-limited settings, dried blood spots represent an easy and suitable alternative to plasma for the determination of HIV-1 viral load. Objective :  To validate a real-time determination of HIV-1 RNA viral load in dried blood spots by comparison with standard plasma viral load.

7. Study design : At the laboratory, patients were asked to authorize the use of their samples for this study and, in this case, they signed a consent. 1 week 82 HIV-1 infected patients Whole blood to get Plasma Whole blood to get DBS Storage of DBS at room temperature Storage of plasma at -70ºC Viral load vs

8. Population n= 82 patients Mean Age38 years (9-55) Mean Hematocrit40 % (25-49) Mean CD4 (total)424 cells/ml (0-1257) Mean CD4 (%)23 % (0-56) On treatment38 (46%) Without treatment44 (54%)

9. Specimen Preparation : We take two whole blood samples in EDTA tubes. Dried blood spots: 6 spots x 50 ul each one. ( Whatman 903 ® Collection Paper) Air dried overnight. Stored at room temperature in specimen collection bags with dessicant during one week. Plasma: 1000 ul. Cryopreserved until being used PLASMATIC Viral Load DBS Viral Load

10.  Cut out the complete blood spots (2 spots of 50 ul per extraction) from the filter using a pair of scissors.  Add the spots into a Nuclisens ® Lysis Buffer 2 ml tube.  Incubate the tubes on a roller mixer for 2 – 3 hours at room temperature.  Centrifugate the tubes during 15 seconds at 1500 g to spin down the fluid.  Transfer the lysate for nucleic acid extraction into a new tube. DBS: Pre-Extraction Procedure

11. RNA isolation (Nuclisens ® miniMag TM)  Addition of an internal calibrator  Incubation with magnetic silica  Washings  Elution Sample + Internal calibrator HIV-1 viral load (Nuclisens ® HIV-1 EasyQ ) At the same time:  Isothermical molecular amplification at 41ºC  Detection of the amplified product by “molecular beacons”

12. Results: 1 copy/ml = 1 IU/ml NMinMaxMeanStd. DevMedianIQ log plasma VL 472.435.74.420.734.300.93 log DBS VL 472.495.74.190.824.281.28 Dif log(Plasma-DBS)47-1.181.10.230.480.290.67 Number of undetectable and detectable cases in both types of specimens

13. Spearman`s Rho n=820.946P<0.001 n=47 VL>50 copies/ml 0.810P<0.01 n=31 VL<50 copies/ml Indetectable by two methods Correlation between both types of specimens 2.0 3.0 5.0 4.0 Log VL DBS 2.03.0 5.06.0 4.0 6.0 Log VL Plasma Bland Altman´s Analysis Averages log plasma VL – log DBS VL

14. Results according to range of values n Plasman DBS <50 IU/ml3135 50-40031 401-300064 >300042 Total82 4 samples >50 in plasma 4 samples <50 in DBS SENSITIVITY: 92.2% SPECIFICITY: 100% POSITIVE PREDICTIVE VALUE: 100% NEGATIVE PREDICTIVE VALUE: 88.6% Copies/ml

15. Conclusions Conclusions: 1.This study demonstrates that DBS performed well as compared to plasma in monitoring viral load. 2.Since no false positive and only 4 false negatives were found (all with VL<3000 copies/ml), we conclude that DBS is a simple and cheaper strategy that may be considered as an alternative to standard VL quantification in resource-limited settings. 3.Although sensitivity was reduced in samples with low – level viremias, DBS would be capable of identifying patients in need of switching to second-line ART.

16. Acknowledgements:  All the patients who contributed with this study  Fogarty AIDS International Training and Research Program  Fundación Huésped  bioMerieux Argentina " We have no solutions in our hands to the world's problems. But to face the world's problems we have our hands." P. Menapace Thank You very much!!!