Winfrida Cheriro1.,2, Gideon Kikuvi2, Simeon Mining3, Wilfred Emonyi3, Erick Rutto3, Elijah Songok4 and Michael Kiptoo4,5*
1Moi Teaching and Referral Hospital (MTRH), Kenya
2omo Kenyatta University of Agriculture and Technology (JKUAT), Kenya
3Moi University School of Medicine (MUSOM), Kenya
4Kenya Medical Research Institute (KEMRI), Kenya
5South Eastern Kenya University (SEKU), Kenya
Received:05 September, 2015;Accepted: 24 September, 2015; Published: 25 September, 2015
Prof. Michael Kiptoo, Principal Research Scientist, 5South Eastern Kenya University (SEKU), Tel: +254722756076; E-mail:
Cheriro W, Kikuvi G, Mining S, Emonyi W, Rutto E, et al. (2015) Drug Resistance Testing in HIV Infected Individuals on Treatment and Naive: Implications on Treatment Outcome. J HIV Clin Scientific Res 2(2): 062-068. DOI: 10.17352/2455-3786.000016
© 2015 Cheriro W, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
HIV/AIDS; Antiretroviral; Drug resistance; Drug resistance mutations
Background: The Government of Kenya started offering ART in the public sector since 2003. Despite the dramatic reduction in AIDS related morbidity and mortality, the emergence and spread of drug resistance (DR) threatens to negatively impact on treatment regimens and compromise efforts to control the epidemic. Therefore, there is a need for information on the situation of DR Mutations (DRMS) and their implications on treatment.
Objectives: To evaluate DRMS and their implications on treatment in HIV infected individuals attending Moi Teaching and Referral Hospital (MTRH) clinics.
Method: In 2009, we consecutively collected plasma samples from two groups of HIV infected individuals, antiretroviral (ARV) naive and ARV experienced for more than 12 months and failing therapy according to world health organisation (WHO) guidelines. We performed genotypic DR using well established in-house Sanger sequencing methods. We then followed up the patients and compared the DRMS in relation to their drug regimens at the time of sample collection and16 months later.
Result: We successfully extracted and sequenced 75 samples. Median age was 36.7 years. Out of 41 drug naive individuals only 3 had DRMS. Out of the 34 ARV experienced, 29 had DRMS to nucleoside reverse transcriptase inhibitor (NRTI), and 31 to non NRTI (NNRTI). After 16 months from sample collection date, 20/31(64%) ARV experienced patients with DRMS had not been changed therapy and only 5/20(25%) were susceptible to primary ARV while 12/14 changed were susceptible to new ARV.
Conclusion: The information obtained in our study can serve as an indicator of ARV program efficiency in patients still on treatment, those who are to start treatment and those who are to be changed therapy due to failure. DR testing would be necessary before initiating and /or changing ART in order to achieve optimal clinical outcome.
AIDS: Acquired immunodeficiency syndrome; AMPATH: Academic Model Providing Access to Health care partnership clinics; ARDR: Antiretroviral Drug resistance; ART: Antiretroviral treatment; ARV: Antiretroviral; AZT: Zidovudine; CD4: Cluster of differentiation; DNA: Deoxyribonucleic acid; DR: Drug resistance; DRMs: Drug resistance mutations; EFV: Efavirenz; HAART: Highly active antiretroviral therapy; HIV: Human immunodeficiency virus; MTRH: Moi Teaching and Referral Hospital; NASCOP: National AIDS control program; NNRTI: Nucleoside reverse transcriptase; NRTI: Nucleotide reverse transcriptase; NVP: Nevirapine; PCR: Polymerase chain reaction; PI: Protease Inhibitors; PR: Protease; RT: Reverse transcriptase; SD:Standard deviation; 3TC: Lamivudine; TDF: Tenofovir; TDR: Transmitted Drug resistance mutations;
Most data concerning Human Immunodeficiency Virus (HIV) non-B subtypes remain controversial. Highly Active Antiretroviral Therapy (HAART) has radically changed the clinical outcome of HIV, leading to decreased mortality and morbidity. Development of HIV drug resistance is inevitable in patients on ART. Increase in antiretroviral therapy (ART) in resource-limited settings (RLS) will successfully reduce HIV-related morbidity and mortality . The increase in ART coverage is expected to lead to an increase in drug-resistant strains among experienced patients. Improved access to alternative combinations of antiretroviral drugs in sub-Saharan Africa is warranted .
As the rollout of ART in Kenya is on the rise, there is a need to monitor the patients on ART . The use of Cluster of Differentiation (CD4) and viral load measurements is important in monitoring HIV patients both immunologically and virologically. Though virological and immunological monitoring is important, there is a need to provide HIVDR testing services for patients who are starting therapy and those who are suspected to be failing treatment before they are switched to a different regimen . A public-health approach based on standardized, affordable drug regimens and limited laboratory monitoring is crucial in scale up efforts. The ever-expanding rollout of antiretroviral therapy in RLS without routine virological monitoring has been accompanied with development of drug resistance that has resulted in limited treatment success. A survey performed in Kampala showed a prevalence of transmitted drug resistance at 8.6% .
In Kenya, availability of ART is increasing. As ART use increases there is mounting evidence suggesting that DR will increase over time . A recent cross-sectional study to determine treatment failure and drug resistance mutations among adults receiving first-line (3TC_d4T/AZT_NVP/EFV) and second-line (3TC/AZT/LPV/r) in Nairobi, Kenya, concluded that the detected accumulated resistance strains due to emergence of HIV drug resistance will continue to be a big challenge . Another study carried out to evaluate treatment success and development of ART drug resistance at the Coast Province General Hospital, Mombasa, Kenya, revealed a high rate of treatment success after short term ART in patients treated at a public provincial hospital detected minority complex drug resistance profiles that were predictive of resistance to currently used second-line NRTIs and NNRTIs regimens . In this article we present detailed data on DRMS from patients who had not started ARV and those who were failing with their implications on therapy. Identifying the relevant DRMS among non-B subtypes will be important for monitoring the evolution and transmission of drug resistance, determination of initial treatment strategies for persons infected with HIV non-subtype B . According to International AIDS Society recommendations (IAS), evaluating susceptibility patterns among non-clade B persons should be a high priority because these viruses are by far the most prevalent world-wide. It is believed that surveillance will maximize the utility of first-line therapy and help minimize the cost of providing ART thereby sustaining current antiretroviral drug programs. The HIVDR testing is important as it gives the clinician accurate information of the most appropriate drug options. With ART scale up, there has been a need for monitoring for development of HIV drug resistance at a population level. Therefore, there is a need for country information on the situation of antiretroviral drug resistance (ARDR) to inform on policy guidelines.
Materials and Methods
The study was conducted at Moi Teaching and Referral Hospital (MTRH), AMPATH (Academic Model Providing Access To Health care partnership clinics), Eldoret, Kenya clinics.
The region includes the expansive Rift Valley, Western and Nyanza provinces, a cumulative population of about 15 million. The hospital is located in Eldoret Town in Uasin-Gishu (UG) County, which forms part of the UG Plateau West of the Great Rift Valley, at an altitude of 2118m above the sea level, latitude 00°30'52”N and longitude 035°17'52”E .
The study was conducted on isolates from patients who were known HIV positive attending the study site and met the selection criteria. During September 2009 and October 2011, patients receiving ARV therapy for at least 12 months and were suspected to be failing according to WHO guidelines were consecutively enrolled. After informed consent was obtained, a standardized questionnaire was administered to assess demographic, epidemiologic, clinical, and treatment information. ART-naive patients were also enrolled during the same period at the same study clinics. Samples from patients who had no history of exposure to ARV drugs and ARV drug naïve status according to a medical chart review and personal interview were collected consecutively.
The study conducted was a hospital-based prospective utilizing isolates from HIV positive patients who met the selection criteria. The study clinics provided ART according to the national guidelines for ART scale-up as recommended by WHO surveillance and monitoring surveys.
Data Collection Tools and Procedures
Sample collection: Remnant blood samples collected for CD4 analysis from ARV naïve were centrifuged and plasma was collected. Remnant samples collected for viral load analysis from ARV experienced patients suspected to be failing therapy clinically were also centrifuged and plasma were collected and stored at −80°C.
HIV DNA extraction: HIV-1 nucleic acid was extracted from 400 µl of plasma using the Nuclisens Easy Mag system (Biomerieux, Canada) following manufacturer's instructions.
Reverse Transcription and polymerase chain reaction: HIV-1 protease (PR) and reverse transcriptase (RT) were bidirectional sequenced with an in-house protocol (8). Briefly, viral RNA was reverse transcribed and amplified according to the manufacturer's directions using the QIAGEN one-step RT-PCR kit (QIAGEN, Canada). The primers used were GaGp1-PR-out.for with a sequence of TGA ARG AIT GYA CTG ARA GRC AGG CTA AT and RT-new-out. Rev of CCT CIT TYT TGC ATA YTT YCC TGT T with nested primers GaGp6-PR-in.for YTC AGA RCA GRC CRG ARC CAA CAG C and RT-new-in.rev GGY TCT TGR TAA ATT TGR TAT GTC CA. All reactions were carried out using standard conditions using GeneAmp PCR System 9700 (ABI) thermocycler.
PCR product purification and sequencing
The PCR products were purified using Multi Screen Separations System as previously described  and diluted to 15 ng/ml for DNA sequencing. Amplicons were sequenced using ABI Prism Big Dye 3.1 Cycle Sequencing System (Applied Bio systems, USA) following manufacturer's instructions.
All the data generated in this study was saved in Microsoft Excel worksheets with a detailed database established to capture all the necessary information. Generated sequences were edited using Bio Edit v 7.0.5B. Aligned fasta files were uploaded to Stanford HIV Drug resistance (http://hivdb6.stanford.edu/asi/deployed/hiv_central.pl?program=hivdb&action=showSequenceForm). Phylogenetic relationships of newly derived viral sequences for comparisons with those of previously reported HIV group M from the Los Alamos database by CLUSTAL W profile alignment was utilized. To improve the accuracy of HIV-1 subtyping, the genotyping tool (http://www.ncbi.nih.gov/projects/genotyping/formpage.cgi) was used and the REGA sub typing tool (http://dbpartners.stanford.edu/RegaSubtyping/) was utilized as needed. Drug resistance mutation and subtype data collected from the Stanford HIV database sequence analysis program were manually input into appropriate excel spreadsheet file, verified and corrections made as needed. Categorical variables were presented in form of frequency tables while continuous variables were mainly summarized using means together with standard deviation and median.
To test significance of skewed continuous variables, Wilcoxon rank sum test was employed. Chi-square test was used to compare the association between categorical variables. Fisher's exact test was also used to compare categorical variables where some cells had expected value of less than 5. Level of significance was set at p < 0.05, with a 95 % confidence interval. All analyses were done using STATA version 11.0.
The study was approved by the Institutional Research and Ethics Committee (IREC) of the Moi University School of Medicine (MUSOM) and MTRH Review Board (IREC):(IREC/2010/06) and AMPATH (RES/STUD/17/2010).
Among the 264 individuals who met the selection criteria, 128 were declared to be ART naïve and 136 had been on ARV for more than 12 months and were failing therapy clinically. Mean age was 37.01 years (SD=12.50). Majority were female 44(60.02%) (Table 1).
Polymerase Chain Reaction and Sequencing outcomes: One hundred and ten (110) samples were successfully amplified. Out of these, 75 samples were successful sequenced and analysed for the presence of drug resistance mutations. Out of 75, ARV experienced individuals failing therapy were 34 and most patients, 25 (73.5%) received 3TC + d4T/AZT + EFV/NVP as first-line treatment. Patients who reported treatment interruption or switch were 9(26.5%). Switch concerned mainly replacement of d4T or AZT by TDF or ABC and only 3(8.8%) had been switched to protease inhibitor (PI) regimens (Tables 1-3).
Drug resistance mutations in ARV naive
Drug resistance mutations were identified in 3/41 (7.3%) of patients and per drug class the values were as follows: 1 for PI and 2 for NRTI and 1 for NNRTI. In 1 patient, multiple mutations against 2 drug classes were seen, suggesting that they were probably not naïve (Table 4). Approximately 3/28(10.7%) of female subjects had DRMS. None of their male counterparts had mutations. There was no statistical difference when male vs. female respondents were compared (p=0.235).
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