1. HIV Diagnostics: New Developments and Challenges Orlando, Florida Feb 28 - Mar 1, 2005 Dried Blood Spots in HIV and HCV Epidemiology and Drug Resistance Testing National HIV & Retrovirology Labs Public Health Agency of Canada Ottawa, Ontario CANADA

2. Dried Blood Spots (DBS) use in HIV serological and nucleic acid testing well-documented & validated simple, robust, inexpensive two approved EIA tests (GSrLAV & OT Vironostika) surveillance, diagnostic, clinical care management

3. DBS - Collection Good !Baaad !

4. Dried Blood Spots (DBS) (antibody elution-GSrLAV EIA) 1. 1/4" disk is punched into an uncoated plate. 2. 200uL of specimen diluent (normal bovine serum / 0.1% Proclinadded and mixed well. 3. eluted o/n at 4 0 C. 4. next day samples are brought to RT and mixed well. 5. Mix 40uLsample into 60uL sp.diluent into coated plate. Rest of assay as per norm.

5. CBC HIV-1 Western BlotINNO-LIA HIV Figure 1. Performance of Cameroon DBS on Confirmatory Tests

6. HIV testing Algorithm DBS HIV-1 screen test (bioMerieux Vironostika HIV-1 EIA) Non-reactive Low Reactive s/co<3 Report NegativeRepeat test Reactive HIV-1 Western Blot (Bio-Rad GS WB) Non-Reactive Reactive Positive Negative Report Positive

7. Hepatitis C testing Algorithm (DBS) HCV screen test (Ortho HCV EIA) Non-reactive Low Reactive s/co<3 Report NegativeRepeat screen test Reactive Supplementary screen Test (Innogenetics HCV Inno-test) Reactive Non-reactive Report Positive

8. Applications of DBS in NHRL Diagnostics Surveillance (Prevalence/Incidence) V.I.D.U.S. (Vancouver Intrav Drug User Study) I-Trak M-Trak (MSM in Montreal) International - Kosovo - Pakistan

9. Molecular Analysis of HIV & HCV from DBS Wash/Elute DBS Purified RNA Elute Bind to silica RT-PCRSequencing HIV HCV

10. Figure 2. HIV-1 Drug Resistance Genotyping on Cameroon DBS (Visible Genetics) and Subtyping (inset)

11. HCV-RT-PCR/Sequencing Core5’NCR PCR RT Phylogenetic comparison (LANL HCV Sequence Database) Sequencing

12. HCV-Genotype Distribution (I-TRAK) high HCV 3a high-risk vs low risk populations ?

13. (26,500) (154,300) (31,000) (16,600) (461,200) Viral Load + + + + + + + - - + - + + - + In House (Nested PCR) Trugene (Bayer) ViroSeq (ABI) Performance of HIV DR Methods (DBS-RNA)

14. + RNase + DNase Nature of Nucleic Acid from DBS Extraction

15. FTA 903 DBS – Viral Load 490,000 FTA 903 DBS – Viral Load 20,000 Effect of Storage Temperature -DBS

16. .3.6.9 1 2 Time (weeks) PCR Amplification Effect of HIGH Humidity/Temp – 903 Paper (45% Relative Humidity and 37°C)

17. PCR Amplification.3.6.9 1 2 Time (weeks) Effect of HIGH Humidity/Temp – FTA Paper (45% Relative Humidity and 37°C)

18. # Successful Amplifications (n=5) Day 1 Day 3 Day 6 Day 9 Day 14 0 1 2 3 4 5 Time Stabilization of DBS-RNA by RNAlater (85% Relative Humidity and 37°C) 903 untreated/no desiccant903 untreated/dessicant 903 treated/no desiccant903 treated/desiccant

19. Conclusion/Future Activities - DBS HIV and HCV serology and NA testing Clinical monitoring – viral load/DR testing Venipuncture vs DBS sequences Limits of DBS to ‘extreme’ conditions “DBS are economical, easy to collect, transport and store. Their ease of use and versatility make DBS an ideal tool for large scale surveillance studies, both domestically and abroad”

21. I-Trak Enhanced surveillance to track HIV- and HCV- associated risk behaviours in injecting drug users (IDU). Cross-sectional design Interviewer administered questionnaire Information collected on:  Demographics  Injecting and non-injecting substance use  Injecting, sexual, testing behaviours DBS collected for HIV & HCV serological testing  To describe changing patterns in the prevalence of HIV and HCV at the national and local level.

22. Conclusions 1.I-Track found 21% of infections involving genotype 3, whereas the database estimates for Canada and the US were 3% and 4% respectively, and Bernier et al. found 14% in Montreal. Bernier et al. also found about 30% each 1a and 1b, whereas I-Track was 72% 1a. This suggests that the observed HCV genotype distribution may be unique to IDU when compared to the general population. 2.Although the numbers are still too low to permit complete statistical interpretation, preliminary univariate analysis suggests that the emergence of genotypes other than 1a in IDU is a relatively new phenomenon. 3.Linking surveillance data to findings of molecular analysis (regional clusters, genotype distribution) will lead to more targeted approaches to prevention, helping those who are most susceptible. 4.DBS are economical, easy to collect, transport and store, and produced quality HCV sequence data for this study. Their ease of use and versatility make DBS an ideal tool for large scale surveillance studies, both here and abroad. 5.HIV sent in about 10% HCV infected.

23.  HIV RNA appears to be preferentially amplified (consistent with plasma)  Commercial sequencing kits are compatible although lack of secondary PCR may be problematic for low viral loads  Similar performance between FTA and 903 under “ideal” conditions  Poorer performance for FTA under elevated temperatures and humidity  Humidity is detrimental to recovery ( desiccant & suitable storage pouches)  Improved recovery by pre-treatment of membrane with RNA stabilizer Conclusions Future Work  Quantitate HIV RNA on DBS by real time PCR  Establish consensus between DBS and plasma derived sequences

24. Genotype Distribution Genotype 1a is the most prevalent genotype across all sites, followed by 3a (based on core region). Other genotypes (non-1a/3a) make up less than 8% of the HCV prevalence among I- Track participants. There was a large regional variation in genotype distribution, although the numbers are still too low to permit complete statistical interpretation.

25. What Next? Need to address differences observed between the core and 5’NCR genetic regions through analysis of other genetic regions (E1, NS5B). The phylogenetic information obtained was often insufficient to achieve statistical significance of clusters. Phylogenetic analysis will be repeated with the inclusion of additional regions as they become available. About 10% of HCV I-Track participants were co-infected with HIV. HIV sequencing (from HCV extracts) is currently underway to characterize the co-infections using phylogenetic methods.

26. Study Design A laboratory investigation of factors influencing the durability of DBS particularly under extreme conditions. Type of membrane Viral Load Storage Temperature Duration of Storage Humidity Sequencing Technology Nature of viral target found on DBS (RNA/DNA)

27. Methods Blood samples were collected from HIV +ve patients (with established viral loads) in 10 ml EDTA vacutainers DBS were prepared by spotting 50  l of blood (903 cards) or 200  l of blood (FTA cards) Viral RNA was extracted using Nuclisens extraction system DBS (903) or ¼ DBS (FTA) were cut into four equal strips and lysis buffer added.

28. Methods RT-PCR was performed using 10  l of isolated viral RNA with “One Step RT-PCR” (Qiagen) Secondary PCR was performed using 4  l of RT-PCR amplicon with “Platinum PCR Supermix” (Invitrogen) Secondary amplicons were sequenced on a LiCor 4200 sequencer using simultaneous bidirectional sequencing following manufactures suggested protocol. Primers (two sets) PR1Forward/RT Protease Inner Reverse (2171-2188 to 2901-2925) RT Outer Forward/3’ Half (2813-2836 to 3506-3536)