1. Using DBS for viral diagnosis in ressources limited setting Senegalese experiences

2. Senegal French Speaking Country ≈ 12 millions inhabitants Median age: 19 year Growing rate: 2.3% Urban population: 42% HIV prevalence - General population 0.7% -Pregnant women: 1.7% - High risk group : > 20%

3. Viral diagnosis Early infants diagnosis –Comparison between DNA PCR Roche 1.5 versus Nuclisens RNA EasyQ 1.2 –Determination of viral load in routine on DBS using Nuclisens EasyQ: an adapted solution for remote areas

4. Early Infants Diagnostics (EID)

5. Introduction Diagnosing HIV infection in infants is difficult. maternal HIV antibodies cross the placenta resulting in serologic tests of the baby that are positive but baby may not be infected. Therefore the usual testing methods applied in the baby are misleading and can not determine the real infection status of the baby before 18 months of age, the period needed for a baby to clear all mother’s antibodies in the blood. Early definitive HIV diagnosis of the baby requires virologic testing.

6. Importance of virological testing –If baby is infected but not tested early and treated it becomes life threatening (around 40% may die before the usual, standard testing is possible). –If the virological testing is performed then HIV-infected infants can get early access to the treatment and care. –Virological testing helps monitor the effectiveness of the PMTCT intervention.

7. Virological testing Polymerase Chain Reaction - PCR which is the diagnostic standard in resource-rich settings but has been too complex and expensive for widespread use in most countries with high HIV prevalence. Is the recommended method for Early Infant Diagnosis (EID) by the WHO and American Academy of Pediatrics, and has excellent sensitivity and specificity. –Whole blood in tubes or on DBS

8. Using DBS - sampling Pricking the heelPricking the toe Pricking the fingerDBS filter card after spotting DBS is a new opportunity for Early Infant Diagnosis: Collecting the blood from the baby is easy – simply prick the heel, toe or finger.

9. Other alternatives – Realtime Detection New approach for performing PCR testing. –It is an equipment that simplifies the steps of extraction, amplification and detection that were described above, resulting in shortening the time required to perform these processes. It has more automated functions. Currently sites in developing countries have Real-time PCR which is used mainly for monitoring patients measuring the HIV RNA level.

10. –Evaluate performances for HIV-1 proviral detection using Roche DNA Amplicor 1.5 versus RNA detection using NucliSens Easy Q® –Compare efficiency and turn around time. Objective

11. Methodology Study Population : –123 Samples → peripheral and regional sites included in PMTCT, or infants with clinical symptoms mainly malnutrition. Types of samples : –43 EDTA Tubes : 5mL transported within 6 h in the lab –80 Paper filter ou DBS

12. Results (1) Population Characterization –123 samples ↔ 101 children – Sexe :34 Girls (33,7%), 55 Boys (54,5%) et NP= 12 –Age : 5 classes median = 4 months 8,90% 5,90% 13,90% 18,80% 3,00% 0,00%1,00% 9,90% 7,90% 5,90% 9,90% 2,00% 0,00% 3,00% 2,00% 4,00% 1,00% 0% 10% 20% 30% 40% 50% %%% [0-M1[[M1-M3[[M3-M6[[M6-M18[>=18NP Classes % GirlBoyNP

13. Results (2) 88,4 74,6 11,6 23,8 0,0 1,6 0,0 20,0 40,0 60,0 80,0 100,0 % de patients 1 sample2 samples3 samples DBS Test 1 Test 2 NucliSens EasyQ PositiveNegativeTotal DNA Proviral Roche® Positive100 negative033 Total103343 Samples frequency Test KHI 2 : p=0,000<0,05 Se : 100,0% Sp : 100,0% PPV : 100,0% NPV : 100,0 Kappa Coefficient : 1.000000 Concordance for samples from EDTA tubes EDTA Plasma

14. Results (3) Test 1 Test 2 NucliSens PositiveNegativeTotal DNA Proviral Roche® Positive140 negative066 Total146680 Test du KHI 2 : p=0,000<0,05 Se : 100,0% Sp : 100,0% PPV : 100,0% NPV : 100,0 Kappa Coefficient : 1.000000 Concordance for samples from DBS The 24 positive samples came from 21 children 90% did not get the PMTCT prophylaxis

15. Discussion Using RNA detection as tool of EID – ROUET et al. (2001) Côte d’Ivoire : quantitation of HIV RNA among breastfeeded children Comparison of RNA/DNA techniques –on EDTA tube Young et al.(2000) : 100% specificity and comparing PCR DNA/RNA –On DBS and tube COMEAU et al. (1996): sensitivity 96% comparing an house DNA PCR ADN on tube and DBS samples. JACOB et al. (2008): sensitivity 95% comparing an house DNA PCR ADN on tube and DBS samples. Turnaround time –NucliSens: simple, easy to perform 3-4 h –Amplicor ADN VIH-1 (ROCHE): turnaround longer 6-8h

16. Conclusions DBS –Useful for early infants diagnosis DNA PCR RNA detection –Equivalent performances Nuclisens EasyQ vs Amplicor DNA 1.5

17. Determination of viral load in routine

18. Background Laboratory tests are essential for –monitoring the efficacy of antivirals –the optimal management of antiretroviral treatment. LTCD4+ count Viral load The measurement of HIV RNA levels in plasma –Several methodologies are available They require adequate facilities and trained personnel, which are available in a limited number of laboratories in developing countries.

19. Antiretroviral drugs are increasingly available in resource- limited countries, –  need to develop more practical and reliable methods that are adapted to field conditions for the collection, transportation and accurate measurement of HIV-1 viral load. Dried blood spots have the potential to further reduce the expense of virological testing by simplifying sample collection, storage and shipment for VL determination. –Proven to be effective for: the detection of HIV-1 DNA (Cassol et al, 1991), quantification of HIV-1 RNA (Alvarez-Muñoz et al., 2005; Brambilla et al., 2003; Cassol et al., 1997; Fiscus et al., 1998; Mwaba et al., 2003a; Uttayamulkul et al., 2005) ), monitoring of the emergence of drug resistance mutations (Plantier and al 2005) Background (cont’d)

20. The present work aimed at : –evaluating the value of dried blood spots for transport and storage of blood specimens collected under field conditions and their use for real-time determination of HIV-1 viral load with NucliSENS EasyQ. –RNA levels obtained with dried blood spots after storage at 37°C for 8 and 15 days were compared with those obtained with plasma from a set of 41 clinical samples collected in suburban dispensaries. –Applying in 7 settings throughout the country Objective

21. Deliverables Determine concordance between –DBS, –blood –plasma viral load results Investigate stability of DBS Investigate reproducibility Applications on the remote areas

22. Methodology Optimisation, Reproducibility, Field Application –Clinical, HIV-1 positive patients –HIV-1 control material (Pelyspy = virions!) –DBS 5 spots (50 µl blood) are applied to individual circles of Guthrie cards (S&S 903 Specimen Collection Paper (Schleicher & Schuell)) by using a micropipettor and plugged tips. –cards were stored in zip bags with desiccant at 37 °C and used for isolation of nucleic acids on day 8 (t = 8) and day 15 (t = 15).

23. Methodology DBS

24. Methodology Study outline Concordance, Stability and Reproducibility (1,8 and 15 days) –Between the HIV-1 viral load results of DBSs and plasma. 20 HIV-1-positive patients’ samples 3 controls whole blood, plasma, and DBS Concordance (8 and 15 days) –41 fields samples Analyses between the methods, the Bland & Altman approach was used, Field Application

25. Results Concordance : blood versus plasma

26. Results Concordance : plasma versus DBS (fresh)

27. Results Stability : DBS

28. Results Reproducibility : DBS Dried blood spots were tested in triplet at each time point.

29. Viral load in plasma vs dried blood spots on day 8 Fig. 1A load results in dried blood spots on day 8 as a function of results for viral load in 100µl plasma (in log10 IU/ml). r = 0.98 Fig. 1B (B) Bland-Altman analysis. The horizontal lines represent the mean difference as well as the mean limits of 2 standard deviations. Coumba Toure Kane, et al 2008 Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal Journal of Virological Methods 148, 291–295

30. Viral load in plasma vs dried blood spots on day 15. Fig. 2A: Viral load results in dried blood spots on day 15 as a function of results for viral load in 100µl plasma (in log IU/ml). In red: the 1st bisector (right Y=X). r = 0.94 Fig. 2B: Bland-Altman analysis. The horizontal lines represent the mean difference as well as the mean limits of 2 standard deviations. Coumba Toure Kane, et al 2008 Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal Journal of Virological Methods 148, 291–295

31. Dried blood spots on day 8 vs dried blood spots on day 15 Fig. 3: (A) viral load results in dried blood spots on day 15 as a function of results for viral load in dried blood spots on day 8 (in log IU/ml). In red: the 1st bisector (right Y=X). r = 0.95 Fig. 3: (B) Bland-Altman analysis. The horizontal lines represent the mean difference as well as the mean limits of 2 standard deviations. Coumba Toure Kane, et al 2008 Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal Journal of Virological Methods 148, 291–295

32. Field application Senegal : Training number of sites –Early Infants Diagnostic n= 8 –Viral load n= 8 –Genotyping n = 8 Sub Region –Bissau Guinea Bandim Project coordinated Statens Serum Institute Copenhagen Denmark Applied sites

33. Field application Senegal –219 patients under HAART since 1 year 74 detectable level > 4log 10 copies/ml 145 under detectable level –Genotyping on the extract is ongoing

34. Conclusions DBS –Showed high concordance, even after two weeks incubation at 37°C. –HIV ‑ 1 quantitation could be used in dried blood testing and hence offers a good alternative for centralized testing in resource-poor countries. –Need to determine the low detectable level –Could be extended on genotyping testing

35. Acknowledgments Senegal team –Coumba Toure-Kane 1, Halimatou Diop Ndiaye 1, Sada Diallo 1, Ibrahima Ndiaye 2, Abdoulaye Sidibé Wade 3, Souleymane Mboup 1 –1Bacteriology-Virology (HALD) –2Infectious disease Clinic (FANN) –3Ministry of Health International partners –bioMérieux (Lyon France, Boxtel Netherland) –BMS Foundation (STF)

36. Thanks for attention