1. Workshop on ART in Pregnancy, Breastfeeding, and Beyond Johannesburg, South Africa June 18-20, 2012 HIV Drug Resistance During Pregnancy and Breastfeeding: Impact of Transmitted HIV DR Resistance in Infants Emilia D. Rivadeneira Pediatric HIV Care and Treatment Team, MCHB, DGHA Centers for Disease Control and Prevention

2. Outline General concepts on HIV Drug Resistance What is known about HIV DR in infancy? What are the programmatic implications and what can we do?

3. General concepts on HIV Drug Resistance Most HIV DR knowledge is based on Subtype B virus studies Pathways to development of resistance may vary by subtype Innate resistance HIV1 vs. HIV2 susceptibility to NVP HIV DR resistance may be primary or secondary – Primary/transmitted resistance refers to resistance acquired through transmission – Secondary/acquired resistance refers to resistance that develops in presence of drug pressure Some mutations confer cross resistance and may affect a whole group of drugs

4. ART have varying resistance thresholds. Drugs with lower thresholds include: NNRTIs and 3TC – NNRTIs in general have a low threshold for resistance: one point mutation in the viral RT gene confers resistance; long ½ life of NVP contributes to viral selection. – M184V is major mutation conferring resistance to 3TC. Combination therapy reduces the risk of resistance development but also may increase the risk for multi-class resistance. Methods to detect major or minor resistance variants have been developed. General concepts on HIV Drug Resistance (cont.)

5. What is known about HIV DR in infancy?

6. Both transmitted and acquired resistance has been described in the pediatric population. Adherence to ART is critical to reduce the risk of resistance. Transmission of resistant virus may occur in utero, intra partum or through breast feeding. ARVs can be detected in breast milk of HIV infected mothers. – Different penetration of ARVs in breast milk may favor the development of resistance and be associated transmission of resistant strains.

7. Infected infants may be exposed to sub-optimal levels of multiple drugs (breast feeding) or monotherapy (ZDV or NVP) for long periods of time before being diagnosed and initiated on treatment. HIV exposed infants are poorly retained in most PMTCT programs to date and less than a third have had access to HIV testing. Sd-NVP has been associated with HIV DR in infants and has lead to changes in the recommendations for 1 st line regimens in this age group.

8. Early evidence of transmitted resistance related to PMTCT expansion Kamkamidze et al. Journal of Clinical Virology, 2001 US Study compares frequency of ZDV resistant strains in infants infected before 1994 vs.infants infected after the widespread use of ZDV for PMTCT. Occurrence of mutations at codon 215. 49 infected infants 12/49 had virus with 215 mutations. 6.3% of infants born between 1992-1994 had mutations in codon 215 vs. 33.3% of infants born between 1998-1996. Increase in trend of ZDV resistance corresponding to increase in maternal ZDV prophylaxis.

9. Prevalence resistance to NVP in mothers and children after single dose exposure to prevent vertical transmission of HIV-1: a meta analysis Arrivé et al. (Journal of International Epidemiology, 2007) Meta-analysis to estimate the proportion of women and children with NVP resistance detected in plasma 4-8 weeks post partum after sd-NVP. – >1244 reports initially found; – Prevalence of DR in infants was estimated from reports from 10 studies conducted between 2000-2006 and corresponded to 15 study arms and a total of 339 infected infants. – 6 studies in southern Africa (SA, Malawi, Uganda, Cote d’Ivoire) – 15 study arms included: 4 maternal AP+IP and Neo sd NVP +ZDV; 1 study arm included an additional dose of NVP post-partum; 2 maternal PP and Neo ZDV+3TC ; 1 arm gave AP/IP/PP ZDV+3TC and neo ZDV and 1 Neo ZDV prophylaxis only

10. High levels of NVP resistance in both women and children. Studies with additional post-partum ARVs had significantly lower NVP resistance rates. – Univariable meta-regression AP, PP and PN ZDV or ZDV+3TC interventions were associated with lower NVP resistance prevalence in infants. – Prevalence of HIV DR to NVP in 52.6% of children in 7 study arms using sd-NVP only and 16.5% in 8 study arms combining sd-NVP with other ARVs. Prevalence resistance to NVP in mothers and children after single dose exposure to prevent vertical transmission of HIV-1: a meta analysis - (cont.)

11. Kisumu Breast feeding Study (KiBS): secondary analysis (Zeh et al, PLos Medicine, 2012) KiBS was a single arm open label PMTCT trial to assess the safety and efficacy of ZDV + 3TC plus Nevirapine or Nelfinavir given from 34 weeks gestation to 6 months post delivery. All infants received sd NVP 522 women (310 NVP arm and 212 on NFV arm) 24 infants were infected by 6 months (15 in the NVP arm and 9 in the NFV) Resistance mutations found at – 2 weeks: 0/8 (six specimens not amplifiable) – 6 weeks: 6/20 (30%) – 14 weeks: 14/22 (63%) – 6 mos: 16/24 (67%) Common mutations: M184V; K103N 9/9 infants of women in the NFV arm and 7/15 NVP had HIV DR

12. Minor resistant variants MacLeod et al, 2010. 33 infants on ART (with NVP) were studied for pre-treatment NVP resistance. – 26 had previous exposure to NVP. Routine sequencing and allele-specific PCR (ASPCR) ViroSeq identified 3/33 with NVP resistance all with treatment failure; ASPCR identified 9 NVP DR mutations in 16 children with treatment failure compared to 4/17 without virologic failure. Low level drug resistance detected in plasma after NVP exposure may be associated with virologic failure on ART. NVP exposed infants <7 months of age were more at risk for developing virologic failure.

13. Prevalence of primary drug resistance associated mutations among vertically infected children in Belo Horizonte, Brazil Ferreira et al. (AIDS Research and Human Retroviruses,2010). ART naïve children, pol gene sequencing in 41 samples stored between 2003-2007. Median age at genotyping was 6 years; 48% CDC stage B; median VL 4.6 log and median CD4 count of 556; median age at first visit was 3.84 yrs. Antepartum maternal ARV exposure for PMTCT in 3 mothers – ( 2: ZDV/3TC/Lpv/r; 1: ZDV monotherapy); Six women received IP ZDV; Neonatal ZDV in 8; Breastfeeding at any time (70.7%) B subtype: 63.4%; F 24.4%; C 2.4% Primary DRM in 4 infants (9.8%): – 3 had DRM associated to RT (M41L; K103N; D76N+ K70R+ M184V+ T215Y+ K103N); – 1 DRM in the protease (L90M)

14. No combined DRM of NNRTI and PI were found The subject with 3 TAMS –D67N, K70R, T215Y- M184V and K103N was exposed to antenatally to ZDV+3TC+Lpv/r; received 6 weeks of ZDV and was not breastfed. Polymorphisms were found in the PR gene in positions 63, 39, 93, 77; in the RT gene: T69S/N/T & A62V/A/P in 2 patients; K211L/E/E26/41. A98G/S in 3 patients and V179D/F/T/I/V in 4 patients. Limited sample size, however with high primary HIV DRM in perinatally infected children in this cohort of children. This finding lead the Brazilian national program to recommend genetic testing for all children prior to ART initiation regardless of age and history of exposure to PMTCT drugs. Prevalence of primary drug resistance associated mutations among vertically infected children in Belo Horizonte, Brazil (cont.)

15. Breast milk transmission and HIV Drug Resistance Accounts for a significant amount of vertical transmission (up to 42%). Possibly higher risk in early breastfeeding period (0.6-0.7%/month between 1-12 mos and 0.3%/mos in the second year of lactation. Variable data on transmission associated with cell associated virus or cell free virus. ART decreases cell free HIV 1 RNA in breast milk Of 10 ARVs (ZDV, 3TC, NVP, LPV, ritonavir, efavirenz, NFV, indinavir, tenofovir, emtricitabine) only ZDV and 3TC are concentrated in BM. – NVP and 3TC plasma levels in BF infants of women on ART - below optimal concentration Several studies have demonstrated the development of DR in breast milk. The prevalence of mutations in breast milk after sd NVP ranges from 37 to 65% 4-12 weeks after delivery and fades with time. Women on cART or with PP ZDV/3TC develop less resistance in breast milk. DR mutations in breast milk can vary between breasts and between plasma and breast milk.

16. Study of plasma and BM isolates of HIV-1 from women on HAART. 26 pregnant women AZT/D4T+ 3TC+ NVP from 28 wks gestation until 1 month post partum. Plasma, whole bm and bm cells were collected and analysed at 1 mos pp. Sequences were isolated from 23 plasma and bm samples and 18 bm cell samples. All strains were subtype C except for one A subtype 4/23 had > mutations in plasma and breast milk (17.4%) > resistance mutations in plasma: K103N + M184V; K103N; V108N; M 184V in plasma; 2 had identical strains in bm -1 in cell free fraction- and 2 had no mutations in bm. 2 women had M184I +M46I (bmc) V106A (cell free fraction) not present in plasma. No significant differences were found between plasma and breastmilk concentrations of ARV detected by LC with UV detection. Breast milk transmission and HIV Drug Resistance (cont) Andreotti et al (AIDS, 2007) – DREAM study-

17. Adherence to combination prophylaxis for PMTCT Kirsten et al. (PLos One, 2011) Observational study in 122 pregnant women starting combination prophylaxis in rural Tanzania -2008-09-. During pregnancy: ZDV from week 28; IP: sdNVP + ZDV +3TC; PP: ZDV + 3TC tail for 7 d Newborn: sd NVP within 72 hrs + ZDV for 7 d (if maternal ZDV >4weeks) or ZDV for 4 weeks (if maternal ZDV <4 weeks) Poor IP adherence: maternal age <24; no income generating activity; and enrolment before 24.5 gestational weeks. Better IP adherence: women who reportedly disclosed their status. IP and PP drug adherence during hospital stay was related to staff performance/levels. – 64 hospital deliveries: 26 women below 80% adherence. Of 63 newborns, 32 ARVs with 80% adherence and 12 at least 95% adherent. Only 10 MIP had 80% adherence during all interventions phases; 1 MIP met 95%.

18. What are the programatic implications and what can we do? Improved follow up of HIV exposed infants and their mothers – Support for adherence from diagnosis – Improved documentation of ARV exposure in infants born to HIV infected women Medical records for HIV exposed infants – Earlier diagnosis of HIV infected infants Optimize first line treatment for infants – Increase availability of PI containing regimens is needed. Improve access to effective monitoring of infants and children on treatment for earlier detection of virologic failure Evaluation of adherence among HIV infected women and their infants – Drug level detection on DBS Population based monitoring of HIV DR in relevant pediatric populations

19. Assessing Transmitted HIV DR in HIV infected infants Bertagnolio et al (CID 2012) Presents a protocol proposed by WHO, HIVResNet and CDC to assess HIV DR in infants Expansion of HIV DNA testing on DBS offers an opportunity to conduct HIV DR surveillance on left over DBS samples from positive infants. Protocol provides – List of require and optional variables to be collected with samples is provided. – List of sample inclusion and exclusion criteria. – Sample size calculations are provided (n=245 to 490 samples) Limitations: – Incomplete data with DBS samples may not allow to test for the association between ARV exposure and HIV DR; – samples may be inappropriately collected, transported, stored leading to over or under estimation of resistance; – coverage of EID may limit generalizability CDC HQ has developed a protocol that is aligned with WHO for implementation in Uganda

20. Finally, Optimize EID sample storage, data Optimized pediatric ARVs and FDCs are still needed to provide effective and acceptable 1 st line regimens for children. – Studies to optimize the dosing of ARVs in very young infants. – Studies in young children with new ARVs or ARV combinations will be needed. – Impact of cART for PMTCT on future 1 st line choices will need to be better understood. More studies on HIV DR development in subtype C viruses is needed. Regional HIV DR databases based on local sequences.

21. Thank you!