1. Partners in Prevention Jim Hughes in collaboration with, and many slides stolen from … Connie Celum Mary Campbell Jai Lingappa Gerry Myers Jim Mullins

2. HIV and Herpes HIV + HSV-2  increased HIV viral loads

3. HIV and Herpes HIV + HSV-2  increased risk of transmitting the virus

4. HIV and Herpes Acyclovir (and related drugs) are very effective in suppressing the HSV-2 virus Acyclovir has no direct effect on HIV but … Acyclovir   HSV-2 replication   HIV viral load   HIV transmission  Can we reduce the risk of HIV transmission in HSV-2 + HIV- positive individuals by treating their HSV-2 infection with acyclovir? ?

5. Partners in Prevention HSV-2 Suppression to Prevent HIV Transmission Acyclovir 400 mg twice dailyPlacebo twice daily 3408 HIV-discordant couples with HIV+ partner also HSV 2-coinfected 1° endpoint: HIV infection in HIV-negative partner Randomize HIV/HSV-2 + partner Follow couples for up to 2 years

6. Nairobi, Thika Eldoret, Kisumu Kenya (4) Kampala, Uganda Moshi, Tanzania Soweto, Orange Farm, Cape Town S. Africa (3) Gaborone, Botswana Lusaka, Kitwe, Ndola, Zambia (3) Kigali, Rwanda

7. Partners in Prevention HIV-infected partner (also HSV-2 infected) = “index” HIV-uninfected partner = “partner” Intervention: Daily acyclovir (400 mg, bid) given to the index Primary outcome: HIV infection measured on the partner – compare infection rates between the two arms Two analyses of interest: i.All transmissions (“easy”) ii.Only transmissions from the index (“hard”)

8. Is the index the source of the partner’s HIV-1 infection? HIV viral sequence is extremely variable due to rapid mutation rate Procedures for determining “linkage” first developed by Gerry Myers, Jim Mullins, Glen Satten and colleagues for the “Florida Dentist Case” in the early 1990’s: Ou, et al 1992. Science 256:1165; Learn and Mullins. 2003. HIV Sequence Compendium 2003:22. Phylogenetic Trees Genetic Distance Amino Acid Signature Pattern

9. Phylogenetics Basic Concepts Trees are comprised of tips, branches and nodes Tips represent the actual gene sequences used to create the tree A branch is a representation of the genetic distance separating tip sequences. A node represents the hypothetical ancestor of the sequences on the branches stemming from it Monophyletic group = Clade terms used interchangeably implies descent from a single common ancestor

10. Simple Phylogenetic Tree branch node root node monophyletic sequence pair tip

11. Genetic Distance Number of mismatched positions Distance = ______________________ Number of positions in alignment the number of nucleotide changes needed to make one sequence the same as another in an alignment can be calculated from pairwise comparisons or estimated by tracing distances between sequences on a phylogram

12. Phylogenetic Linkage Analysis 3.3% 13.4%

13. Is the index the source of the partner’s HIV-1 infection? Monophyletic pair Gives yes/no answer, but no degree of uncertainty Should intervening sequence automatically rule out linkage? Should lack of an intervening sequence automatically confirm linkage? Genetic Distance “Linked” partners should have sequences that are close; unlinked partners should have sequences that are not close Is there a “magic” distance which separates linked and unlinked? Not quite but …

14. Smoothed probabilities for distances between sequence pairs known to be linked or unlinked Distance linked unlinked 0.00.10.20.30.4

15. Bayesian Classification of partner linkage D = distance P(D | linked) = probability of distance D assuming linkage (based on intra- individual sequence pairs and partners known to be linked) P(D | not linked) = probability of distance D assuming no linkage (based on sequence pairs from apparently unlinked individuals) P(linked) = assumed probability of linkage before looking at D. P(linked | D) = posterior probability of linkage; base decision on this.

16. Issues/Challenges At present, the algorithm is based on distances only. In principle, additional quantitative data could be incorporated into the information D. What should the prior – P(linked) – be?

17. Issues/Challenges At present, the algorithm is based on distances only. In principle, additional quantitative data could be incorporated into the information D. What should the prior – P(linked) – be?  We set the prior equal to the proportion of linked partnerships over all PIP seroconverters  Most decisions not very sensitive to choice of prior

18. Issues/Challenges Classification may be based on individual sequence pairs (multiple per partnership) or consensus sequence pairs (generally one per partnership) Single consensus sequence may not represent all variants Combining information from multiple sequence pairs poses methodologic challenges The transmitting partner may have multiple variants but only one may match the variant found in the newly infected partner. Should this be considered linked? What if the situation is reversed?

19. Pair 58

20. Contrasts between Bayesian and phylogenetic approaches  Bayesian classifier based on distances only and does not explicitly consider context (i.e. monophyletic or not)  In this application, monophyletic approach tends to more conservative (more likely to call the sequences unlinked); more appropriate for courtroom?  Use of explicit prior assumption in Bayesian formulation allows control of “conservativeness”  Bayesian classifier can incorporate additional information on partnership (e.g. gender, reported number of sex partners) in through prior

21. Questions What scientific question is the PIP trial trying to answer? Why is it difficult to to determine if one person has transmitted the HIV virus to another (even if you have virus samples from both individuals)? What genetic measurement does the Bayesian classifier use to determine the probability of transmission?