dc.description.abstract | Despite the efficacy of antiretroviral therapy (ART) and the advancement in the prognosis of
persons living with HIV/AIDS, a considerable proportion of persons on ART do not realize or
retain adequate virologic suppression. Currently, 68% of adults and 73% of children living with
HIV in Kenya are receiving ART treatment following the implementation of the 2015 World
Health Organization (WHO) test and treat guidelines. During this study, the first-line ART
treatment recommendations in Kenya typically comprised of two nucleoside reverse transcriptase
inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI): either
nevirapine (NVP) or efavirenz (EFV). High interpersonal variability in the pharmacokinetics of
ARV drugs have been reported, which may jeopardize ART treatment gains if not appropriately
managed. Therapeutic drug monitoring (TDM) of antiretrovirals (ARVs) purposes to identify
elevated or sub-therapeutic ARV concentrations which allows for prompt dosage adjustments
preventing patients’ exposure to toxic or subtherapeutic concentrations. Optimal ART outcomes
inevitably require an understanding of the individual variation in response to ART. This study
determined the pharmacogenetic and pharmacoecological determinant of sub-therapeutic
responses to NVP and EFV among HIV patients in Nairobi Kenya.
In this non-comparative cross-sectional study, a total of 599 HIV patients were recruited who
provided 5ml blood samples 8 hours post ART medication as well as participated in a face to face
structured interview. The CD4 cell, viral load, full blood hematology and blood chemistry
measurements were determined according to manufactures’ instruction. HIV drug resistant
mutations were identified using an in-house sequencing method while cytochrome P450
(CYP26B) and constitutive androstane receptor (CAR) single nucleotide polymorphisms (SNPs)
were identified using Real Time Polymerase chain reaction (RT-PCR). The NVP and EFV plasma
levels were measured using ultra-high-performance liquid chromatography with a tandem
quadruple mass spectrometer (LC/MS/MS). All data were subjected to descriptive statistical
analysis. The NVP and EFV plasma levels were first tested for normality and presented as the
median and interquartile range (IQR). Inferential data analysis of drug plasma levels were
evaluated using the student’s t-test and one-way ANOVA. Categorical variables were summarized
as frequencies and percentages while the Pearson’s chi square and fisher-exact tests used for
inferential analysis of categorical variables. Host factors relate to NVP and EFV plasma level
either directly or indirectly by affecting ART adherence. The relationship between
pharmacoecological factors with ART adherence was first evaluated using fisher-exact or chisquare
and only significant variables were evaluated for association with NVP or EFV plasma
level using quantile linear regression. Data analysis was done using STATA v 13 software
(StataCorp LP, Texas, USA). The allele, genotype and haplotype frequency and deviation from
Hardy-Weinberg of CYP26B and CAR single nucleotide polymorphism (SNP) was analyzed using
an online SNPStats software (https://www.snpstats.net/start.htm). The level of significant was set
at p<0.05.
All the 599 patients enrolled (100% responses rate) had data for objective one, while 566 (94.5%
responses rate) had data for objectives two, three and four. The median age of the 599 patients was
41 years [IQR 35-47 years] with the majority (60.3%) being female and 56.1% were receiving
EFV based regimen. The CD4 cell count (mean ± SD) significantly increased at the12-month post
ART initiation (301.7 ± 199.4 cell/ml to 329.4 ± 305.8 cell/ml; P<0.05). Hepatotoxicity and renal
abnormalities occurred more frequently at month 12 compared to baseline; ALT (2.5% versus
10.5%), AST (5.3% vs 23.4%) and creatinine (63.4 vs 68.84%). Fewer patients at month 12 had
anemia (29.4% vs 56.4%), leucopenia (42.4% vs. 46.9%) and thrombocytopenia (6.5% vs. 84.1%)
compared to baseline. The median [interquartile range – IQR] NVP plasma (n = 254) concentration
was 6237.5 [4518 – 8964 ng/ml] and 2739.5 [1878 - 4891.5 ng/m] for EFV (n = 312). Majority
54.3% of patients had supra-therapeutic NVP plasma levels followed by 31.5% and 14.2% with
sub-therapeutic and therapeutic plasma concentrations respectively (p < 0.001). Patients on EFV
(63.8%, 31.7% and 4.5%) had therapeutic, supra-therapeutic and sub-therapeutic plasma
concentrations respectively (p < 0.001).
Thirteen CYP2B6 (329G>T, 341T>C, 444 G>T/C, 15582C>T, 516G>T, 548T>G, 637T>C,
785A>G, 18492C>T, 835G>C, 1459C>T and 21563C>T) and one CAR (540C>T) SNPs were
detected among study patients. Hardy-Weinberg equilibrium could not be tested for CYP2B6
329G>T, 341T>C, 444 G>T/, 637T>C, 835G>C, and 548T>G SNPs due to lack of heterozygous
and/or homozygous mutants. Linkage disequilibrium (LD) was observed among CYP2B6
15582C>T, 516G>T, 785A>G, 18492C>T, 983T>C, 21563C>T, 1459C>T and CAR 540C>T,
resulting in 8 haplotypes among which CTGCTTCC and CGATTCCT had the highest and the
lowest frequency. The most frequently occurring mutations with a prevalence of more than 30%
were CYP2B6 516G>T, 785A>G and 21563C>T followed by CYP2B6 18492C>T and 15582C>T
at a frequency of between 10 to 20%. The CAR (540C>T) and CYP2B6 983T>C occurred at a
rate of 5 to 10% with CYP2B6 1459C>T, 329G>T, 341T>C, 444 G>T/C, 637T>C and 835G>C
rarely occurred (>0.05%). The genotype and allele frequencies of SNPs were similar, regardless
of the ART regimen. The following SNPs: CYP2B6 329 G>T; 15582C>T, 516G>T, 785A>G and
21563C>T, were associated with reduced NVP metabolism; while CYP2B6 18492C>T and
983T>C with increased NVP metabolism. The SNPs CYP2B6 15582C>T, 516G>T, 785A>G, and
21563C>T were associated with reduced metabolism of EFV and CYP2B6 18492C>T with
increased EFV metabolism. The CYP2B6 and CAR inferred phenotypes associated with a reduced
metabolism were found in 171/566 (30.2%) of all study patients; and 91/312 (29.2%) on EFV and
80/254 (31.5%) on NVP. Phenotypes associated with increased metabolism were found in 39.9%
(n = 226/566) of the patients, 122/312 (39.1%) patients on EFV and 104/254 (40.9%) patients on
NVP.
On multivariate quantile regression analysis, factors that remained associated with high NVP
plasma concentration included; feeling guilty for being HIV positive (adjusted β 954, 95% CI
192.7 to 2156.6; p =0.014) or feeling worthless for being HIV positive (adjusted β 852, 95% CI
64.3 to 1639.7; p =0.034), disclosing patient’s HIV status to neighbors (adjusted β 1731, 95% CI
376 to 3086; p = 0.012), regular uptake of porridge (adjusted β 1780, 95% CI 121.4 to 3438.6; p
=0.036), missing taking current ARVs for the whole day or more (adjusted β 4287.9, 95% CI,
826.2 to 7749.6; p = 0.015), current white blood cell concentration (adjusted β 4012.4, 95% CI
1032.2 to 6992.6; p < 0.001). The SNPs associated with NVP plasma levels on multivariate
analysis included; CYP2B6 329G>T (adjusted β 11343.2, 95% CI 5738.8 to 16947.6; p <0.001),
CYP2B6 341T>C (adjusted β -8614.2, 95% CI -9621.7 to -7606.7; p < 0.001), CYP2B6 516G>T
(adjusted β 3603.4, 95% CI 589.6 to 6617.2; p = 0.019), CYP2B6 18492 C >T (adjusted β -1301.4,
95% CI -2195.3 to -407.5; p = 0.004), CYP2B6 983T>C (adjusted β 2948, 95% CI 313.2 to 5582.8;
p =0.028), CYP2B6 CAR 540 C>T (adjusted β -1543.2, 95% CI -2683.7 to -402.7; p =0.008) and
number of SNPs per patients (adjusted β 1129.6, 95% CI 729.9 to 1529.3; p <0.001).
On multivariate quantile regression analysis factors that remained significantly associated with
high EFV plasma levels included; disclosing HIV positive status (adjusted β 363, 95% CI, 97.9 to
628.1; p = 0.007), obtaining ARV pill uptake information from other sources (adjusted β 18421.3,
95% CI, 16291.5 to 20551.2; p <0.001), stopping taking current ARVs due nausea (adjusted β 713,
95% CI 425.5 to 1000.5; p <0.001) and stopped taking current medication due to skin problems
such as rash (adjusted β 1847, 95% CI 137.9 to 3556.1; p =0.035) and the presence of body pain
in the past 30 days (adjusted β 475, 95% CI, 117.2 to 832.8; p = 0.009). The SNPs CYP2B6
516G>T (adjusted β 2414, 95% CI 1876.6 to 2951.4; p <0.001), CYP2B6 835 G >C (adjusted β
2877, 95% CI 1498 to 4256; p <0.001) and number of SNPs per patient (adjusted β 570, 95% CI
362 to 778; p <0.001).
Some of the important limitations worth mentioning in this study included. First, the use of NVPbased
ART regimens in Kenya and other countries, especially developed countries, has been
considerably reduced in the recent past, meaning that this study could be relevant to a restricted
number of patients. Second, standardized tools for measuring pharmacoecological variables are
not available, limiting the generalizability of this study outcomes. Third, this was a cross-sectional
study, which only permitted the description of the relationship between pharmacogenetic and
pharmacoecological factors and NVP/EFV plasma concentrations and not a causal conclusion.
Such outcomes can be confirmed in a longitudinal study. These limitations notwithstanding, the
following conclusions can be drawn. These data establish prolonged immunologic/virologic
response to ART among patients continuing on therapy. The prevalence of anemia,
thrombocytopenia and leucopenia was low with minimal renal and hepatotoxicity impairment
observed. Wide interindividual variability were observed in NVP and EFV plasma concentrations
with a large proportion of patients falling outside of therapeutic window, proposing potentially an
increased risk of treatment failure or toxicity. This study demonstrates the importance of
therapeutic drug monitoring in determining the ARV treatment outcome. Six SNPs of
the CYP2B6 gene (CYP2B6 329G>T, CYP2B6 637T>C, CYP2B6 785A>G, CYP2B6 18492C>T,
CYP2B6 21563C>T and 15582C>T) and one CAR (540T>C) could potentially act as additional
independent predictors of NVP and EFV plasma concentrations beyond that provided by
the CYP2B6 c.516G>T and CYP2B6 983T>C polymorphism. Host pharmacoecological factors
influence ART drug adherence impacting on the NVP and EFV plasma concentrations. | en_US |