Phylogenetic relationships of HIV-1 Pol RT strains Multi-drug Resistant HIV-1 among Kenyan Patients on Long-Term Highly Active Antiretroviral Therapy Timothy Nzomo1,2, Rose Kitawi1,2, Ruth Sada1,2, Rashid Aman1,3, Joyceline Kinyua4, Bernhards Ogutu1,4,6 Florence Oloo,1,6,7, Gilbert K’Okwaro1,6 and Washingtone Ochieng1,4,5,6 1. Center for Research in Therapeutic Sciences, Strathmore University ; 2. Institute of Tropical Medicine and Infectious Diseases; 3. African Center for Clinical Trials; 4. Kenya Medical Research Institute; 5. Harvard University School of Public Health. 6. Institute of Healthcare Management 7. Technical University of Kenya Abstract To assess the prevalence of Drug resistance-associated mutations (DRAMS), we sequenced the pol-RT region of HIV-1 in 54 patients enrolled at various treatment centers in Kenya. At least 27.8% of the patients harbored DRAMs, with 73.3% of the mutations conferring resistance to both nucleoside RTIs (NRTIs) and non-nucleoside RTIs (NNRTIs). Of all DRAMS, 47% were NRTI-specific while 52.8% were NNRTI-specific. Another 81.7% of patients with NNRTI DRAMs had high-level resistance to more than one drug. All patients with NRTI DRAMS had high-level resistance to at least 3 different ARVs. The most common of NRTI and NNRTI mutations were M184V/I and K103N occurring respectively in 73.3% and 60% of the patients with DRAMs. Background The RT gene undergoes constant mutations in the absence or presence of drugs If mutations occur on sites targeted by drugs, resistance may develop These sites include; the pol-RT, pol-PR and pol-IN The emergence of drug resistance mutations (DRMs) to the major classes of available anti-HIV drugs poses major threats to treatment. Objective In this study we evaluated ARV drug resistance genotypes from a cross-section of HIV-1 infected subjects receiving care and treatment from various treatment centers across Kenya Methodology Results Table 1: Reverse Transcriptase-Inhibitor Resistance Mutations in Cell-free Blood Fifteen of 54 (27.8%) harbored DRAMs. NRTI DRAMS (47%). NNRTI DRAMs (52.8%) M184V (73.3%) K103N (60%) Table 2: Frequency of DRAMs and Drugs to which they confer Resistance Phylogenetic relationships of HIV-1 Pol RT strains Patient ID Subtype ARV Regimen NRTI mutations NNRTI mutations KAH004 A1D TDF+3TC+NVP D67G, K70E, M184I V90I, K101E, Y181C, G190A KHC093 A1 D4T+3TC+NVP None K103N KMB077 D M184V V90I, K103N KMB160 AZT+3TC+NVP D67N, K70R, M184V, K219Q Y188L, G190A MLD003 NR E138A MLD011 MLD013 D67N, K70R, M184V G190A MLD016 MLD040 A1C V108I, Y181C, H221Y MLD060 C K103N, V106M MLD183 ABC+3TC+NVP L74V, Y115F, M184V K103N, H221Y, F227L MLD191 V90I, K101E, E138G MLD198 A1A2 M41L, V75I, M184V, T215Y MLD245 A2B MLD545 M41L, M184V, T215F K103S, E138Q NRTI DRAM Selected drug N (%) NNRTI DRAM M184V/I 3TC, FTC. 11 (44) K103N/S NVP, EFV 9 (32.1) D67N/G AZT, D4T, ABC, TDF, DDI 3 (12) E138A/Q ETR, RPV 3 (10.7) K70R/E TDF, ABC, DDI & 3TC, FTC G190A T215Y/F D4T, ABC, DDI, 2 (8) V90I +All NNRTIs M41L AZT H221Y 2 (7.1) K219Q AZT & D4T 1 (4) K101E NPV, EFV, ETR, RPV L74V ABC, DDI Y181C Y115F ABC F227L 1 (3.6) V75I D4T, DDI V106M V108I Y188L NVP, EFV, RPV Total 25 (100) 28 (100) A phylogenetic tree of pol-RT sequences. A total of 54 isolates with sequences covering HIV-1 pol-RT region are shown. Bootstrap values of 50% were used . Subtype A1 was the most prevalent (57.4%). Other subtypes were D (14.8%), C (5.6%) and recombinantins (22.2% ) Patient consenting, enrollment and blood-draw (April-Aug 2013). DNA obtained from PBMCs (Qiagen Qiamp DNA Blood Mini Kit) RNA obtained from blood plasma (Qiamp viral RNA Mini Kit) Reverse transcription (Qiagen One Step RT PCR Kit) PCR amplification using gene specific primers: Pol-RT region (nt 2480-1380 HXB2) Purification of PCR products (Qiagen PCR Purification Kit) Sequencing (Macrogen, Amsterdam) Sequence analysis Manual editing &annotation(BioEdit). Alignment- ClustalW Drug Resistance mutations Inference Stanford HIV DR Database & Geno-to-Pheno Phylogenetic Analysis Using Mega 5.1 Kimura 2-parameter with 1000 bootstraps Extensive use of Nevirapine and Efavirenz, preceding the appearance of K103N mutations contributed to high-level resistance and cross-resistance to other NNRT drugs. Subtype A viruses were the most prevalent (57.4%), with recombinants also present in a significant proportion of samples (22.2%) This observation is consistent with other findings in Kenya (1,2) Recombinant viruses had the highest average of DRAMs per subtype at 4.5. Recommendations Drug-resistance testing should be integrated to monitoring programs to improve treatment efficacy Results Summary & Discussion All patients with NRTI DRAMs had high-level resistance to at least 3 different ARVs. Up to 81.7% of patients with NNRTI DRAMs had high-level resistance to more than 1 drug The NRTI mutation M184V/I was detected in a vast majority of patients 11/54 (73%), in combination with other TAMs This was the reason for the high-level resistance to 3TC and FTC observed in all specimens with the mutation, and the low-level cross resistance to DDI observed in 9 patients Conclusion We demonstrate that a significant proportion of patients undergoing treatment have developed multiple drug resistance Our data suggests a near universal resistance to NVP and EFV, that are commonly and alternately used in this setting. DRAMs affecting 3TC also dampened the efficacy of ART Acknowledgement This work was supported by the Consortium for National Health Research (CNHR), Kenya, with funds from the department for International Development and the Welcome Trust (UK). Grant #RCDG005 to Dr. W. Ochieng References 1. Kitawi ,et al. AIDS Res Hum Retroviruses. 2014, 30 (0),2014 2. Lihana, et al. AIDS Res Hum Retroviruses 2009,25:1211–1217.