2. Source: EPP/Spectrum HIV estimates, EPHI, 2014

3. HAART is the recommended form of treatment HAART is usually combination of at least 3 drugs 2 NRTIs + 1 NNRTI or 1-2 PIs

4. Standard ART regimens Restricted drug options Limited lab monitoring Decentralized service delivery and task shifting People receiving ART 12.9 million globally 11.7 million in LMIC 38% of people living with HIV

5. Success ART coverage reached 75.4% in 2013 ART made available in both HCs & HPs since August 2006 # ever started ART increased from 900 at the beginning of 2005 to 743,299 by June 2013. ART sites rose from 3 in 2005 to 913 by 2013.

6. Rapid scale up is prone to emergency and transmission of HIVDR It is very important to remain watchful! Tracking of transmitted HIV DR is an Tracking of transmitted HIV DR is an expensive business High laboratory costs WHO recommends minimum-resource method to evaluate transmitted drug-resistant HIV: Threshold Survey (HIVDR-TS) Small sample size < 50 And yet reliable results Can be done periodically

7. HIVDR Threshold Surveys The HIV Drug Resistance Threshold Survey (HIVDR-TS) : The HIV Drug Resistance Threshold Survey (HIVDR-TS) : Method supports classification of the prevalence of transmitted HIV drug resistance in a specific geographic area into 1 of 3 categories: LOW prevalence < 5% LOW prevalence : < 5% MODERATEprevalence5% and 15% MODERATE prevalence : Between 5% and 15% HIGH prevalence > 15% HIGH prevalence : > 15% – Method doesn’t estimate point prevalence – Result not national or clinic specific but apply to the geographical area surveyed within the country

8. Pooled analysis from 82 surveys covering a total of 3588 recently-infected patient -72 surveys with results that could be classified as low, moderate or high moderate levels of resistance to any drug class -20 (28%) reported moderate levels of resistance to any drug class Moderate levels of resistance :- - Moderate levels of resistance :- 18% in 2004-2006 period to 32% in 2007-2010 Transmitted HIVDR (WHO surveys 2004-2010)

9. Prevalence of HIVDR in ARV-naïve individuals, since ARV rollout Gupta et al. Lancet 2012

10. undertake HIV drug resistance threshold survey in Gondar University Hospital and Gondar health center To undertake HIV drug resistance threshold survey in HIV Counseling and Testing clients at Gondar University Hospital and Gondar health center

11. Study design Cross-sectional study Study site Selection Based on WHO recommended criterion Gondar University hospital and Gondar health center Greater risk factors for emergence of HIVDR Holly water Higher number of ART clients Longer period of ART use Previous HIVDR report etc..

12. Study participants VCT clients at GUH and Gondar HCs VCT clients at GUH and Gondar HCs Inclusion criteria Age between 18-25 and no previous pregnancy if female Has lived in the Gondar town for about a minimum of a year Documented positive HIV test result No previous positive HIV test No known exposure to antiretroviral drugs No known AIDS-defining illness Not eligible to start ART First risk-defining event within the past 3 years (e.g.,drug injection, STI

13. DATA COLLECTCTION, MANAGEMENT & PROCESSING

14. VCT center Check eligibility Consent participants Draw blood (10ml) Send participant data to database Test specimen for HIV Send sample to lab Laboratory Run CD4 count Prepare DBS Separate plasma Fill Sample transmittal form Store samples Send ST form to database Database Check Survey codes for identity Enter data into database Lock hard copies in a cabinet Ship data to EHNRI along with samples EPHI Receive specimens based the specimen receipt SOP Synchronize data with existing database Store specimens at -80 Data samples DATA FLOW

15. HIVDR report Sequence data editing Automated Sequencing Purification Cycle sequencing Purification Gel electrophoresis Amplification RNA extraction QIAamp Viral RNA mini kit Six primer (3 forward, 3, reverse ) QIAquick purification kit 1% agarose gel electrophorosis RT-PCR & Nested PCR Stanford HIVDR database Recall ABI 3100/3500xl Genetic analyzer Isopropanol

16. 06-99 PR 1-251RT RT-PCR PrtM-F1 RT-R1 Nested PCR Prt-F2 RT-R2 3 reverse primers AV44,90V1, RT-R2 3 forwarded primer A35V, AV36V, Prt-F2 Forwarded primer Position HXB2 1. Prm-F1 2057-2085 2 Prt-F2 2243-2266 3. A35V 2556-2577 4. AV36V 2869-2889 Reverse primer Position HXB2 1. RT-R1 3370-3348 2. RT-R2 3326-3304 3. AV44 2952-2931 4. 90V1 2639-2619 CYCLE SEQUENCING RT-PCR & Nested PCR 06-99 PR1-251RT

17. Transmitted HIV-1 Drug Resistance Analysis TDR mutations – Calibrated population resistance (CPR) tool version 6.0 (http://cpr.stanford.edu/cpr/servlet/CPR )http://cpr.stanford.edu/cpr/servlet/CPR Rates and frequencies of ARV drug resistance associated mutations – Stanford Genotypic Resistance Interpretation Algorithm http://hivdb.stanford.edu/pages/algs/HIVdb.html http://hivdb.stanford.edu/pages/algs/HIVdb.html  Phylogenetic analysis was done using MEGA 5 & BioEdit (Version 7.0.9.0)

18. Study Population Follows the sequential sampling method selected by WHO for the surveillance of transmitted HIVDR in low-resource settings A total of 84 samples were collected 64 from Gondar university hospital 20 from Gondar health center The mean age of participants was 21 years (range: 18–24 years) 70 (83.3%) were females

19. Sample Number genotyped (SNG) Lower Limit (LL) Running total of specimens with HIVDR (RT) Upper Limit (UL) Sample Number genotyped (SNG) Lower Limit (LL) Running Total of specimens with HIVDR (RT) Upper Limit (UL) 1ND0 25ND06 2 0 26ND06 3 0 27NDK103N 6 4 0 28NDG190S 6 5 0 29ND2 6 6 0 30ND2 6 7 0 31NDK103N 6 8 0 32ND36 9 0 33ND36 10ND0 34136 11ND0 35137 12ND0 36137 13ND0 37137 14ND0538137 15ND0539137 16ND0540137 17ND0541137 18ND0542137 19ND0543137 20ND0544237 21ND0545237 22ND0546238 23ND0547238 24ND05STOP

20. WHO HIVDR threshold category 67 (78%) 67 (78%) were successfully amplified and sequenced WHO TS binomial sequential sampling method. HIVDR prevalence was categorized using the WHO TS binomial sequential sampling method. 3 specimens were carrying NNRTI resistance mutations (K103N G190S, K103N) after genotyping of 47 sequences According to the WHO recommendations of sampling and classification plan prevalence of transmitted HIVDR is classified as – Moderate prevalence (5% - 15%)

21. Prevalence of transmitted HIVDR for each class of ARV moderate (5%–15%) NNRTI moderate (5%–15%) low (<5%) NRTI low (<5%) low (<5%) PI low (<5%) By analysis of the 67 samples amplified and sequenced – 4(6%) samples with major HIVDR mutation associated with NNRTI – No NRTI and PI associated major mutation

22. HIVDR among treatment naïve patient in Gondar town Year of sample collectionOverallNNRTINRTIPI Kassu et.al 20033/92= 3.3%G190A (n=2)V75I ( n=1)No Andargachew 2008/098/155=5.2%Y188HY (n=1) K101E (n=1) G190A (n=2) K219E (n=1) L210W (n=2) K65R (n=1) No Dawit et al 2011/124/67= 6%K103N (n=2) G190S (n=1) Y181Y (n=1) NoneNo

23. Our results indicated that the levels of TDR to be increasing, Moderate prevalence (5%–15% ). Overall 6.0% prevalence of primary HIVDR An increasing ARV drug exposure in populations, following the roll-out of ARVs for treatment and prevention of mother-to child transmission, may cause a rise in TDR in the study area.

24. CountryYearPrevalenceYearPrevalence Zambia2005O%200915.3% Uganda2006/70%2009/108.6% Cameron20011.9%200712.3% Kenya20057.5%2009/1013.2%

25. Similar reports In the Africa region, a more detailed analysis by drug class showed a statistically significant increase in the prevalence of NNRTI mutations. 36% East Africa 36% per year 23% Southern Africa 23% per year 5.1% Estimated prevalence of NNRTI resistance was 5.1% 8 years after rollout in east Africa. WHO HIVDR report 2102

26. DRMs among untreated HIV-1-infected The current results and others clearly showed an increase in prevalence of DRMs among untreated HIV-1-infected individuals in Gondar town repetition of the survey Needs to be confirmed through repetition of the survey in this area and possibly in additional areas of the country. possible sources of HIVDR transmission Furthermore work should be done to identify possible sources of HIVDR transmission,