Molecular characterization of HIV drug resistance mutations in protease and reverse transcriptase genes in treatment naïve and experienced patients in care in Eldoret, Kenya

Abstract

The Government of Kenya started offering ARV Therapy in public sector since 2003. Kenya is one of the six human immunodeficiency virus (HIV) ‗high burden‘ countries in Africa. Kenya HIV Estimates Report in 2014 indicated that, nationally over 1.6 million patients were living with HIV and over 600,000 patients were receiving antiretroviral (ARV) Therapy (ART) with the national HIV prevalence at 7.6% in women and 5.6% in men. The use of ARV has resulted in reduction in acquired immunodeficiency syndrome (AIDS) related morbidity and mortality but led to emergence and spread of ARV drug resistance (DR) (ARVDR) which threatens to negatively impact on treatment regimens and compromise efforts to control the epidemic. Sanger sequencing of non-clonal Polymerase Chain Reaction (PCR) amplicons of plasma viral cDNA is widely used to detect drug resistance mutations (DRMs) in the molecular targets of HIV-1 namely reverse transcriptase (RT) and protease (PR) genes. A major limitation however, is its inability to detect low abundance of DR viruses (LADRVs) existing in a patient‘s plasma sample which have been shown by several studies to be clinically relevant often leading to failure of new ARTs. The use of next generation sequencing (NGS) has been shown to be more sensitive for LADRVS. The main objective of this study was to characterize inter subtype RT and PR gene mutations of viral isolates obtained from HIV infected ARV naive and ARV experienced patients failing therapy according to WHO guidelines. From October 2009 to October 2011, patients who met selection criteria were consecutively enrolled in this study through Moi Teaching and Referral Hospital (MTRH), Kenya. A total of 206 participants aged 6.6 years to 71.8 years were included in the study. Primers were specifically designed to amplify PR and RT. HIV-1 nucleic acid was extracted from plasma samples, reverse transcribed, amplified then sequenced to determine DR using Sanger sequencing and NGS, the new 454 pyrosequencing technologies. The mean age was 38.1 (SD: 12.2) years, 62.9% were female, 50.9% were married, engaged or cohabiting, 42.0% had secondary or tertiary level of education. Of the study participants 32.5% were ARV naïve and 52.5% in WHO clinical stage 3 or 4. There were 15 (7.6%) participants who reported opportunistic infections, 8 (4.0%) had tuberculosis, and 8 (4.4%) had sexually transmitted diseases. The median CD4 cell count per cubic milliliter was 210.0 (IQR: 70.0, 391.2) and the average viral load (log 10) was 3.1 (SD: 1.5), range (1.6 – 5.7). Of the 206 participants, 114 (55.3%) had their samples successfully amplified samples went through both sequencing techniques. A total of 83 participants were successfully sequenced using Sanger method. Up to 59.0% were ARV naïve. The average age was 36.9 (SD: 12.2) years with a range of 6.6 – 66.2 years. Sixty percent were female, 54.3% were married, and 42.0% had a secondary or a tertiary level of education From successfully edited sequences, 49 were from ARV naïve while 34 were from ARV experienced, 39.8 %( 33/83) were male, mean age was 36. Among ARV naïve, 3/49 (6.1%) who were female patients were identified with DRMs of which, one had PI, two NRTI and 1 had NNRTI. None of their male counterparts had mutations. From the ARV experienced, Mean age was 35.85years (SD=14.06). Male were 18(52.9%). Most, 67.2% received 3TC + d4T/AZT + EFV/NVP as first-line treatment with 32.7% having EFV and 60% having NVP in their drug regimen. Those who reported treatment interruption or switch were 32.7%; mainly replacement of d4T or AZT by TDF or ABC and only 7% had been switched to protease inhibitor (PI) regimens. Subtype distribution were as follows; A 22(65%), A/D 2(6%), A/K 1(3%), AE 1(3%), B/A 1 (3%), D 3(9%), D/A 3(9%) and G 1(3%). Using the xxiii new 454 pyrosequencing approach, 60 samples from ARV naïve were successfully sequenced of which 25 were subtype A, 11 subtype D, 1 Subtype C and the remaining were recombinants whereby,46 (76.6%) had at least one DRM; with 25 (41.6%) indicated as major and the rest 21 (35%) indicated as minor. The most prevalent mutation was NRTI position K219Q/R (11/46, (24%)) followed by NRTI M184V (5/46, (11%)) and NNRTI K103N (4/46, (9%)). The use of NGS technology in this study revealed a high prevalence of LADRVs among drug naive populations in Eldoret Kenya. The information obtained in this study can serve as an indicator of ARV program efficiency. DR testing would be necessary before initiating and /or changing ART in order to achieve optimal clinical outcome. DRMS were identified in ARV naïve patients with a prevalence of 6.1%. All naïve patients identified with DRMs were female. The most prevalent mutations identified in ARV naïve patients were those affecting NRTI 2/3(67%). DRMS were identified in ARV experienced patients with a prevalence of 6.1%. The most prevalent NRTI mutation observed was at position M184IV while the most prevalent NNRTI mutation observed was at position K103N. Drug Resistant Mutations across gender was statistically significant. Overall, all Male subjects from ARV experienced had DMRS when compared to Females. In this study a high prevalence of 41.6% of LADRVs among drug naive populations was revealed. Drug Resistance testing would be necessary before initiating ARV therapy so as to guide in the choice of susceptible combination ARV. It is highly recommended to use a feasible next generation sequencing technologies for surveillance of HIV drug resistance at population level to reliably detect and monitor emerging drug resistance patterns that may impact ARV treatment. There will be a need for a continued follow-up of persons with DRMS and LADRVS to determine clinical impact and help guide therapies for drug naïve populations