1. 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 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), Lihana, et al. AIDS Res Hum Retroviruses 2009,25:1211–1217.