Effect of pharmacogenetics on plasma lumefantrine pharmacokinetics and malaria treatment outcome in pregnant women

Considering anticipated population proportion (P) of clinical failures in pregnant women being 18% [19], with 95% confidence level and 10% precision, 92 malaria positive pregnant women were enrolled in this study.

The study participants received six doses of four tablets of ALu (Coartem^®; Novartis Pharma AG, Basel, Switzerland) (20 mg artemether and 120 mg lumefantrine) over the course of 3 days at 0, 8, 24, 36, 48, and 60 h. For each patient, general physical examination was performed at enrollment (day 0) and on follow up visits on days 2, 7, 14, 21 and 28. Approximately 50 μl of blood was collected on filter paper (Whatman grade 3) for later PCR analysis. Each filter paper was dried and individually stored in a plastic bag and kept frozen at −80 °C. At enrollment day, 1 ml of whole blood was taken into an EDTA containing vacutainer tube and stored at −80 °C at MUHAS laboratory until further analysis. Additionally, 3 mls of venous blood were drawn from pregnant women in heparinized tubes on day 7 to determine plasma LF concentrations.

Genomic DNA was isolated from peripheral leukocytes in whole blood samples using QIAamp DNA Midi Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Genotyping for the common functional variant alleles for CYP2B6*6, CYP2B6*18, CYP3A4*1B, CYP3A5*3, CYP3A5*6, CYP3A5*7 and ABCB1 c.4036A>G (rs3842), which have been reported to be relevant for artemether and LF disposition [26, 27] were done as described previously [12, 22]. In brief genotyping was performed using TaqMan drug metabolism genotyping assay reagents for allelic discrimination (Applied Biosystems Genotyping Assays) with the following ID numbers for each SNP: C__7817765_60 for CYP2B6*6 (c.516G4T, rs3745274), C__60732328_20 for CYP2B6*18 (c.983T4C, rs28399499), C__26201809_30 for CYP3A5*3 (c.6986A4G, rs776746), C__30203950_10 for CYP3A5*6 (g.14690G4A,rs10264272) and C__32287188_10 for CYP3A5*7 (g.27131_27132insT rs41303343) and C__11711730_20) for ABCB1 c.4036A>G (rs3842), and C__11711730_20 for CYP3A4*1B (−392A>G, rs2740574). Genotyping was carried out using Quant Studio 12 K Flex Real-Time PCR system (Life Technologies Holding, Singapore, Singapore). The final volume for each reaction was 10 μl, consisting of TaqMan fast advanced master mix (Applied Biosystems, Waltham, MA, USA), TaqMan 20X drug metabolism genotyping assays mix (Applied Biosystems) and genomic DNA. The PCR profile consisted of an initial step at 60 °C for 30 s, hold stage at 95 °C for 10 min and PCR stage for 40 cycles step 1 with 95 °C for 15 and step 2 with 60 °C for 1 min and after read stage with 60 °C for 30 s.

Dried blood spots on filter papers obtained at enrolment (day 0) and on follow-up days (day 2, 7, 14, 21 and 28) were punched, and one circle 5 mm in diameter was used for DNA extraction using QIAamp DNA blood micro kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s recommendations. Detection of Plasmodium parasites was performed using a species-specific PCR targeting the ssRNA gene [28]. PCRs were carried out in duplicate in a 25-μl final volume containing 12.5 μl of Universal PCR Master Mix, 5 μl of DNA, forward and reverse primers at various concentrations, and P. falciparum, Plasmodium malariae, Plasmodium vivax and Plasmodium ovale probes at a final concentration of 100 nM. All reactions were run on an ABI Prism 7000 sequence detection system (Applied Biosystems) with the default settings; each sample was initially denatured at 95 °C for 10 min and cycled 40 times, with each cycle consisting of 95 °C for 15 s and 60 °C for 60 s. Each reaction plate included four positive controls for P. falciparum, P. ovale, P. vivax, and P. malariae and a negative control with molecular-grade water in place of DNA.

PCR genotyping to differentiate recurrent P. falciparum infections from reinfections were done according to World Health Organization (WHO) recommendations [29], by characterizing the length polymorphism of the merozoite surface protein 2 gene (msp2) in samples collected at day 0 and on the day recurrent parasitaemia was found. Recrudescence was determined when at least one msp2 allele of the same allelic type and of identical base pair size was found in samples collected on day 0 and on the day of recurrent infection. A reinfection was defined as all alleles were of different length in the sample collected on day 0 and that collected on the day of recurrent infection.

Blood samples for the determination of LF plasma concentrations were collected on day 7 (corresponding to 168 h) following initiation of ALu treatment. Blood samples for quantification of LF levels were centrifuged and plasma was stored at −80 °C until analysis. Plasma LF concentration was analysed using a validated method of high performance liquid chromatography (HPLC) with ultraviolet detection at Sida/MUHAS bioanalytical laboratory in Dar-es-Salaam, Tanzania [30]. The coefficients of variation (CV %) during the analysis of LF were 8.4, 4.7 and 4.5% at 100, 1000, and 8000 ng/ml, respectively. The lower limit of quantification was 50 ng/ml.

LF plasma concentration data were log transformed to achieve normality of data distribution. Median (interquartile range) was used to describe day-7 LF plasma concentrations. Comparison of day-7 median LF plasma concentrations in pregnant women between the different genotypes were carried out using Kruskal–Wallis one-way analysis of variance (ANOVA). X² test was used to compare the observed and expected allele frequencies according to the Hardy–Weinberg equilibrium. Influence of human genotype on malaria treatment outcome was analysed using Pearson’s Chi square and Fisher’s exact test. Haploview software package (version 4.2) was used to analyse linkage disequilibrium (LD) and haplotype construction. Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software, version 22.0 (IBM Corporation, Somers, NY, USA). P values <0.05 were considered to be statistically significant.

Malaria treatment outcomes were classified following the WHO protocol [31], as adequate clinical and parasitological response (ACPR), corrected for reinfection using PCR genotyping on day 28 and treatment failure (TF); designated as early treatment failure (ETF), late clinical failure (LCF), or late parasitological failure (LPF). The influence of genetic variations on malaria treatment outcome was evaluated using Cox regression analysis.

In total, 1835 pregnant women were screened using MRDT and a total of 92 pregnant women with malaria infection consented and were enrolled in the study. Baseline characteristics are presented in Table 1. The median age of pregnant patients was 23 (range 15–41) years and the majority (54.3%) were multigravida. Most participants were in the second trimester (60.7%). The median parasite density was 2700 (range 400–72,500) parasites/µl.

In total, 424 samples were screened for presence of parasites and P. falciparum was present in 11 (12%) patients during the follow up period. Two samples were detected on day 7, four on day 14, one on day 21 and four on day 28 after ALu treatment. PCR based genotyping of msp2 confirmed recrudescent infection in seven women (63.3%), two on day 7, three on day 14, one on day 21 and one on day 28. The remaining four (36.4%) pregnant women had reinfection; one on day 14 and three on day 28. The overall rate of reinfection was 4.3% (95% CI 0.15–8.45).

PCR uncorrected ACPR on day 28 was 88.9% (95% CI 82.06–95.74) and TF rates were 11.1% (95% CI 4.26–17.94). Rate of ACPR was calculated using per-protocol method, where patients were excluded due to lost to follow-up, protocol violations and TF due to reinfection. PCR-corrected ACPR rate as defined by absence of parasitaemia on day 28, irrespective of axillary temperature, in patients who did not previously meet any of the criteria of ETF, LCF or LPF was 91.0% (95% CI 82.62–95.58). The rate of PCR-corrected LPF, as defined by presence of parasitaemia on any day between day 7 and 28 in patients who did not previously meet any of the criteria of ETF or LPF, was 9.0% (95% CI 4.42–17.38). There was no ETF or LCF.

The overall CYP2B6*6, CYP2B6*18, CYP3A4*1B, CYP3A5*3, CYP3A5*6, CYP3A5*7, ABCB1 c.4036A>G genotype and allele frequencies in Tanzanian pregnant women is presented in Table 2. There was no significant deviation between the observed and expected genotype frequencies from Hardy–Weinberg equilibrium. The variant allele frequency was highest (76.1%) for CYP3A4*1B followed by CYP2B6 c.516G>T (*6, 33.5%). CYP2B6 c.983T>C (*18) had the lowest allele frequency which was 9.3%. Genotype of CYP450 enzymes and ABCB1 transporter was equally distributed among the age, gravida, trimester and PMTCT status of pregnant women (P > 0.05).

Haplotype analysis of CYP3A4*1B (–392A>G), CYP3A5*3 (g.6986A>G), CYP3A5*6 (g.14690G>A), CYP3A5*7 (27131_27132insT) is presented in Fig. 1 and Table 3. There was no linkage between the two CYP2B6 variant alleles (c.516G>T and c.983T>C). Likewise, no LD between the three CYP3A5 SNPs (CYP3A5*3, CYP3A5*6, CYP3A5*7) was found. Instead each SNP was inversely linked to each other (i.e., each SNP is in strong LD with the wild type variant of the other two SNPs). The haplotype frequency of CYP3A5*1, *3, *6 and *7 was 44.5, 22.6, 20.7 and 12.2%, respectively. Interestingly all the three CYP3A5 variant alleles (CYP3A5*3, CYP3A5*6 and *7) occur in high LD with CYP3A4*1B. The major CYP3A haplotype was CYP3A4*1B alone (34.2%) followed by its linkage with CYP3A5*6 (17.6%) and CYP3A5*3 (13.3%) (Table 3).

Based on the haplotype analysis result, subjects were grouped according to the numbers of functional CYP2B6*1 and CYP3A5*1 variant alleles; two (*1/*1), one (heterozygous for defective variant alleles) and zero (homozygous for defective variant alleles) to investigate the effect of genotype on day 7 plasma LF concentrations.

The median day 7 plasma LF concentration was 650 with the range of 2936 (124–3059) ng/ml. Comparison of the median plasma LF concentrations between the different genotypes is presented in Table 4. There was no significant effect of CYP2B6, CYP3A4*1B or ABCB1 c.4036AG genotype on day 7 LF plasma concentrations. The median plasma LF concentration was significantly associated with CYP3A5 genotypes (P = 0.039). CYP3A5 defective allele carriers displayed a higher plasma LF concentrations compared to those homozygous for CYP3A5*1/*1 genotypes (P = 0.04). There was significant association between having CYP3A5 genotype and having plasma LF concentration ≥600 ng/ml (Pearson Chi square test, P = 0.01). The proportion of subjects with two functional CYP3A5 genotype (*1/*1) was significantly higher among those with plasma LF conc <600 ng/ml (34.6%) compared to those with ≥600 ng/ml (5.9%). On the other hand proportion of subjects with lacking functional CYP3A5 (homozygous for defective variant alleles) was higher among those with plasma LF conc ≥600 ng/ml (38.2%) compared to those with >600 ng/ml (19.2%).

The possible influence of genetic variations in CYP2B6, CYP3A5 and ABCB1 on malaria treatment outcome was evaluated using Cox regression analysis. A log rank test was run to determine differences in the cumulative hazard distribution between the different genotypes. There were no associations between malaria treatment outcome and ABCB1 transporter and most of CYP450 genotypes with an exception to CYP3A4 (Table 5).