Human Genetic Variation Is Associated With Plasmodium falciparum Drug Resistance

Abstract

One approach to investigate if human genetic variation influences the selection of Plasmodium falciparum drug resistance is to compare the frequency of resistant infections among human populations differing in their genetic background and living in the same epidemiological context. A further complementary approach consists in comparing drug resistance among subjects differing for genes involved in drug metabolism. Here we report, from malariological surveys performed in Burkina Faso, that the prevalence of P. falciparum chloroquine-resistant infections (pfcrt 76T and/or pfmdr1 86Y alleles) differs among sympatric ethnic groups, being higher in the Mossi and Rimaibé groups than in the Fulani group (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.27–3.92; P = .007). The association analysis revealed that the human CYP2C8*2 variant, known to determine a poor drug metabolizer phenotype, was associated with P. falciparum chloroquine-resistant infections (OR, 1.66; 95% CI, 1.13–2.43; P = .008). This variant is more frequent in the Mossi-Rimaibé group (23.7% ± 1.4%) than in the Fulani group (9.9% ± 2.5%; P = .0003). This study provides an example of how host genetic variation may influence the selection dynamics of a pathogen’s drug resistance.

Drug resistance is a leading problem in the control of infectious diseases. Several factors, such as drug usage, transmission intensity, host immune status, and pharmacokinetics, may influence its selection. It is possible that human genetic variation represents another cofactor. In the case of Plasmodium falciparum malaria, it is known that the level of acquired immunity affects the clearance of drug-resistant infections [1, 2]. Because the level of antimalaria immune reactivity, besides cumulative exposure, is determined by genetic factors [3], differences in the genetic predisposition to select for drug resistance among human subjects and/or populations differing in their immune reactivity could be hypothesized. Furthermore, genetic-based variations in drug metabolism may influence drug clearance, inducing different conditions of parasite-drug exposure (ie, time and concentration) and possibly affecting priming for drug resistance [4, 5].

A possible populational approach to investigate this topic is to compare the prevalence of drug-resistant infections among sympatric ethnic groups differing in their genetic-based immune reactivity to malaria and equally exposed to malaria transmission. A further, complementary, strategy is to test the risk of selecting parasite drug resistance according to variation in specific genes involved in drug metabolism.

Almost all drugs developed against P. falciparum are metabolized in the human liver through ≥1 cytochrome P450 enzymes [6]. Genetic polymorphisms of these enzymes determine distinct metabolic phenotypes: poor metabolizer (PM) phenotypes accumulate higher plasma concentrations of drugs and show an increased elimination half-life, experiencing adverse side effects; on the other hand, extensive or ultrarapid metabolizers show lower plasma levels from a standard drug dose [7].

Chloroquine (CQ), one of the most used antimalarial drug, is metabolized mainly by cytochrome P450 2C8 enzyme (CYP2C8) but also by CYP3A4 and CYP2D6 [8, 9]. In humans, CQ concentrations decline multiexponentially. After administration, ∼50% of the dose is excreted unchanged in urine. In the liver CQ is rapidly dealkylated via CYP450 enzymes into the pharmacologically active monodesethylchloroquine. Further desethylation leads to bisdesethylchloroquine (far less active as antimalarial), and minor metabolites are formed by oxidative deamination of the side chain. Monodesethylchloroquine and bisdesethylchloroquine concentrations reach 30% and 2% of CQ concentration, respectively [10, 11]. Variants exist in the 3 known genes (CYP2C8, CYP3A4, and CYP2D6) conferring a PM phenotype. Some of these variants are found at high frequency in sub-Saharan Africa where, as a consequence, adverse side effects such as pruritus are common and associated with a lower metabolic ratio of the drug [12]. CYP2C8 is a member of the human CYP2C enzyme–encoding gene family [13]. This enzyme plays a major role in CQ and amodiaquine (AQ) metabolism and a minor role in that of dapsone (DDS) [13, 14]. In a recent investigation, Parikh et al [15] found that CYP2C8*2, a defective allele, due to a Ile→Phe substitution at position 269 (I269F) [13], shows 6-fold lower intrinsic clearance for AQ than the wild type. This gene variant may have therefore important implications as to malaria treatment and possibly as to the selection of parasite drug resistance. Higher frequencies of this allelic variant are observed in African populations than in whites. In Zanzibar, CYP2C8*2 has a frequency of 13.9% [16], reaching 16.7% in Northern Ghana [17]. A study from Burkina Faso revealed an allele frequency of 11.5% among subjects treated with AQ monotherapy [15]. The frequency of the CYP2C8*2 allele is 18% in African Americans [18], whereas in whites it is very low [13].

As to the minor route of CQ metabolism, available data indicate the existence of a genetic CYP3A4 “African” polymorphism with functional importance as to enzyme expression [19]. The CYP3A4*1B allele is associated with a significantly higher quinine metabolic ratio (lower quinine hydroxylation capacity) than CYP3A4*1 wild-type allele in Tanzanians [19]. As to CYP2D6, the enzyme does not seem to contribute significantly to CQ metabolism due to the relatively small proportion (2%) of the general CYPs content in the liver [20] and the ability of CQ to inhibit CYP2D6-mediated metabolism in vitro and in vivo [9]. Mutations in CYP450 conferring a PM phenotype cause a longer drug half-life, thereby resulting in a longer parasite exposure to a subtherapeutic level of the drug [5]. In addition, there is evidence that the rate of P. falciparum resistant alleles is higher in subjects with residual CQ concentrations in plasma [21].

In this context, to investigate the possible contribution of human genetic variation in the selection of antimalarial drug resistance, we performed a comparative analysis of P. falciparum CQ resistance among 3 sympatric West African ethnic groups—Mossi, Rimaibé, and Fulani—differing in their genetic background [22, 23], susceptibility, and immune reactivity to malaria [24–26] and equally exposed to malaria transmission levels [27]. In particular, we evaluated in the 3 ethnic groups, in a period of initial selection of CQ resistance in Burkina Faso [28], the following 2 parameters: (1) prevalence of CQ-resistant infections (P. falciparum pfcrt K76T and/or pfmdr1 N86Y polymorphisms) [29–31] and (2) frequency of the human cytochrome P450 CYP2C8*2 allele. Moreover, to evaluate the possible relationship between the cytochrome P450 CYP2C8*2 allele and the risk of harboring a P. falciparum CQ-resistant infection, we performed an association analysis in the 3 ethnic groups.

MATERIALS AND METHODS

Study Area and Subjects

The samples analyzed in the present study were collected during a cross-sectional malaria survey performed in August 1994 in a rural area 35 km northeast of Ouagadougou, Burkina Faso [25]. Oral informed consent was obtained for multiple immunoparasitological, genetic, clinical, and entomological surveys. Very intense P. falciparum transmission is recorded during the June–October rainy season, frequently reaching mean sporozoite inoculation rates well above 1 infective bite per person per night. The main malaria vectors are Anopheles gambiae, Anopheles arabiensis, and Anopheles funestus. Information was collected for each ethnic group on movements out of the villages; use of mosquito coils, bed nets, or other mosquito-protective methods; access to healthcare; and self-medication. With particular reference to the use of antimalaria drugs, an ad hoc survey looking at the presence of CQ and its metabolites in urine [32] was performed in the study area during the same 1994 high malaria transmission season. This survey, conducted on a total of randomly selected subsample of 240 subjects (60 Fulani, 93 Mossi, and 87 Rimaibé) did not reveal differences in CQ consumption among the 3 ethnic groups (P = .94).

For the present genetic investigation, finding were analyzed in a total of 506 P. falciparum–infected subjects aged >10 years [71 Fulani, 248 Mossi, and 187 Rimaibé; mean age ± standard deviation, 24.0 ± 15.3, 29.4 ± 16.4, and 27.1 ± 15.2 years, respectively). A 5-mL venous blood sample was collected from each subject. Thick and thin blood smears were prepared according to the World Health Organization guidelines for the microscopic diagnosis of malaria (Bench Aids for the Diagnosis of Human Malaria; plate 8), and 100 microscopic fields (∼20 leukocytes per field at ×1000 magnification, corresponding to ∼0.25 μL of blood) of the thick blood smear were examined. The Plasmodium species was identified on the thin blood smear. Each smear was evaluated independently by 2 expert microscopists. Any discordance was resolved by a third microscopist. Patients with positive smears were treated according to the guidelines of the Ministry of Health of Burkina Faso.

Genotyping

Parasite DNA was extracted with Chelex-100 resin (Bio-Rad) [33]. DNA samples were amplified by nested polymerase chain reaction–restricted fragment length polymorphism (PCR-RFLP) technique to identify K76T point mutations in the CQ-resistant marker pfcrt (on chromosome 7) and N86Y point mutation in pfmdr1 (on chromosome 5), according to a protocol already available [29]. Appropriate P. falciparum controls (3D7 for wild-type CQ-sensitive strains and K1 for CQ-resistant strains) were used for each PCR analysis.

Human DNA was extracted with Chelex-100 resin (Bio-Rad) [33]. CYP2C8*2 (rs11572103, A > T) detection was carried out with a PCR-RFLP technique. We used 2 μL of DNA template to amplify by PCR a 107–base pair (bp) fragment of the CYP2C8 gene (forward primer, 5′-GAACACCAAGCATCACTGGA-3′; reverse primer, 5′-GAAATCAAAATACTGATCTGTTGC-3′). The PCR product was then incubated with BclI enzyme that cuts the wild-type allele only (A); undigested products represent the variant allele (T). To detect the size polymorphisms we analyze the samples using 3% MetaPhor gel. Controls for human genotyping were used after sequencing of PCR product obtained from each different genotype.

To estimate the allele frequency of CYP3A4*1B (rs2740574, A > G), we analyzed the “African” PM alleles for several drugs, including quinine [19], in 41 unrelated subjects (21 Mossi and 20 Fulani). We used 2 μL of DNA template to amplify by PCR a 223-bp fragment of the CYP3A4 gene (forward primer, 5′-CTGGGTTTGGAAGGATGTGT-3′; reverse primer, 5′-TGTTACTGGGGAGTCCAAGG-3′), and then we sequenced the PCR products to identify the A to G polymorphism at position 392.

Statistical Analysis

P values of the comparisons were obtained by Yates-corrected χ² test. Unadjusted odds ratios (ORs) were calculated with 95% confidence intervals (CIs) and are shown in Table 3. Mantel–Haenszel (M–H) weighted ORs and maximum likelihood estimate of ORs (both with 95% CIs) were also calculated after stratification by age (11–20, >20–30, and >30 years) and ethnic group (Fulani, Mossi, and Rimaibé). Each parasite isolate was classified based on the presence or absence of resistance-associated alleles, and infections with mixed wild-type/mutant alleles of pfcrt or pfmdr1 were treated as mutants. To test the association between human CYP2C8*2-carrier genotypes and parasite drug-resistant markers, we created a binary categorization for parasite polymorphisms: CQ sensitive, that is, an isolate containing only wild-type CQ-sensitive allele for both markers; and CQ resistant, that is, an isolate containing the mutant genotypes for one or both markers (thus, including in this category the double-mutant isolates). We used binary logistic regression analysis to test the influence of different independent variables on the dichotomous variable CQ-sensitive/CQ-resistant isolate, calculating ORs and their corresponding 95% CIs. Linkage disequilibrium analysis (in the sense of Adagu and Warhurst [34]) on pfcrt and pfmdr1 genotype association was carried out using GenePop Web software, version 4.0 [35].

RESULTS

P. falciparum CQ Resistance in Fulani, Mossi, and Rimaibé Groups

The overall frequencies (3 ethnic groups combined) of pfcrt-76T– and/or pfmdr1-86Y–resistant infections (mixed infections plus pure mutant infections) were 11.1% ± 1.4% and 37% ± 2.1%, respectively. An inverse association between age and malaria-resistant infections was recorded. The frequency of resistant infections (mixed infections plus pure mutant infections) was 49.5% ± 3.4% in the 10–20-year age group, 38.9% ± 4.7% in the 21–30-year age group, and 35.7% ± 3.7% among those aged >30 years (P = .016). As shown in Table 1, interethnic differences in the frequencies of pfmdr1 (N86Y) genotypes were observed, whereas no significant differences occurred for pfcrt (K76T). Overall, the Mossi and Rimaibé group showed a higher prevalence of CQ-resistant infections than the Fulani group (196/435 [45.1%] vs 21/71 [29.6%]; M-H weighted OR, 2.24 (95% CI, 1.27–3.92); P = .007). To evaluate the possible confounding effect of parasite density, a binary logistic regression analysis was performed. This analysis excluded any effect of parasite density on the different frequencies of CQ-resistant infections among the 3 ethnic groups (OR, 0.99; 95% CI, 0.83–1.17; P = .89). Moreover, we observed an association between the pfcrt-76T allele on chromosome 7 and pfmdr1-86Y on chromosome 5 (χ² = 6.12, P = .013; r² = 0.0082, P = .041), indicating that in this study there is linkage disequilibrium (in the sense of Adagu and Warhurst [34]) between the 2 drug-resistant alleles, possibly maintained through the selective pressure of CQ.

Human Cytochrome P450 CYP2C8*2 (rs11572103, A>T) Allele in 3 Ethnic Groups

As shown in Table 2, the frequency of the CYP2C8*2 (T) allele in the Fulani group was lower than in the Mossi and Rimaibé groups, being, respectively, 9.9% ± 2.5%, 24.2% ± 1.9%, and 23.0% ± 2.2% (P = .0003). The Mossi and Rimaibé groups showed, respectively, 4.8% and 6.4% of individuals homozygous for the T allele, whereas no homozygous allele was recorded among the Fulani (P = .001). The fraction of the population carrying ≥1 T allele was 19.7%, 43.5%, and 39.6% in the Fulani, Mossi, and Rimaibé groups, respectively. The locus was in Hardy-Weinberg equilibrium in all 3 ethnic groups (Fulani, χ² = 1.10, P = .29; Mossi, χ² = 0.35, P = .55; Rimaibé χ² = 0.00, P = 1.00).

Human Cytochrome P450 CYP3A4*1B (rs2740574, A>G) Allele in Unrelated Subjects

The analysis of CYP3A4*1B (G) allele in a subgroup of unrelated subjects reveals an overall frequency of 0.793 without significant differences between Mossi (0.857 ± 0.054) and Fulani (0.725 ± 0.071; χ² = 2.18, P = .14). Thus, the majority of the examined subjects (90.3%) carry the defective allele in the enzyme that constitutes the alternative route for CQ metabolism.

P. falciparum CQ Resistance and CYP2C8 Genotypes

To investigate whether the CYP2C8*2 (T) variant affects the risk of being infected by P. falciparum CQ-resistant strains, we estimated the prevalence of CQ-resistant infections according to the CYP2C8 genotype. As shown in Table 3, trends for a higher risk of carrying pfcrt-76T and pfmdr1-86Y CQ-resistant P. falciparum, in subjects carrying the CYP2C8*2 (T) allele, were observed in all 3 ethnic groups (χ²_trend = 5.45; P = .020). Considering the whole sample, the presence of the T allele was associated with a higher risk of harboring a P. falciparum CQ-resistant infection (OR, 1.66; 95% CI, 1.13–2.43; P = .008). Then we performed a binary logistic regression analysis: the dependent dichotomous variable “parasite CQ resistance” was tested against the independent variables of age, ethnic group, log parasite density, and CYP2C8*2-containing genotypes. After removal of the nonsignificant term (log parasite density; P = .89), the results showed that CQ resistance is associated with CYP2C8*2 after adjustment for age and ethnic group (OR, 1.59; 95% CI, 1.10–2.32; P = .014).

DISCUSSION

Previous comparative studies have demonstrated the existence of genetic-based interethnic differences in the susceptibility and immune response to P. falciparum malaria among West African sympatric ethnic groups with different genetic backgrounds [22–26, 36]. This interethnic comparative approach constitutes an appropriate model to study the role of human genetic variation in host-parasite relationships. In the present study, this model was exploited to investigate whether human genetic variation affects the selection of parasite drug resistance. The Fulani, known to have a genetically based higher immune reactivity to malaria [24–26, 37, 38], showed a lower rate of drug-resistant infections. This evidence is consistent with previous indications showing that the capacity to clear P. falciparum drug-resistant infections is proportional to the level of acquired antimalaria immunity [1]. Consistently, an inverse association between age, that is, cumulative exposure and hence antimalaria immunity, and prevalence of drug resistance, was observed in the present study.

Another indication of the possible contribution of human genetic variation in influencing the selection of drug resistance derives from the observed association between the defective CYP2C8*2 variant and an increased risk of harboring a P. falciparum CQ-resistant infection. The fact that the frequency of this mutation is ∼2-fold lower in the Fulani than in the Mossi-Rimaibé group, together with the genetic-based higher immune-reactivity of Fulani, indicates the existence of interethnic differences in the predisposition to select for parasite drug resistance.

Drug metabolism is essential to establish the half-life of a drug. Watkins and colleagues have shown that the length of the terminal elimination half-life is an important determinant of the propensity for an antimalarial to select for resistance [39–41]. This could be particularly relevant when dosing and levels of compliance with antimalarial therapies are low.

Another interesting result of the study is the higher rate of pfmdr1 than pfcrt resistant genotypes. This could be due to the time of spread of pfcrt-76T allele in West Africa. From the literature, we know that the Asian pfcrt haplotype CVIET moved to East Africa in the late 1970s and throughout Africa in the 1980s–1990s and is now widespread all over the continent [42, 43]. One possible scenario suggested by these data is that the pfmdr1-86Y allele was already present at an appreciable frequency before the spread of resistant pfcrt-76T alleles in Africa after multiple origins of pfmdr1 polymorphisms [44, 45]. Our study seems to confirm the evidence of a prior selection of the pfmdr1-86Y allele in Burkina Faso at the time of the surveys. Although pfcrt is the most important player in CQ resistance, in some epidemiological settings [46–48] and at the onset of resistance [49], pfmdr1 seems to contribute directly to CQ resistance. A finer spatial and temporal resolution of this polymorphism within regional areas of sub-Saharan Africa could help clarify the evolutionary history of CQ resistance.

In general, the finding of this study suggest that the macrogeographic distribution of P. falciparum drug resistance, classically considered the result of drug pressure and transmission intensity, could have been also influenced by human genetic variation. This, in line with the findings of Gouagna et al [50], provides a further example on how host genetic variation, besides influencing resistance or susceptibility to infectious diseases, may affect further interfaces of host-pathogen relationships.

Notes

Acknowledgments.

 We are particularly grateful to the villagers of Barkoundouba and Barkoumbilen for their collaborative attitude throughout the investigation.

Financial support.

 The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant 242095 (Towards the establishment of a permanent European Virtual Institute dedicated to Malaria Research [EVIMalaR]). The work was also supported by the Istituto Pasteur–Fondazione Cenci Bolognetti, University of Rome “La Sapienza.”

Potential conflicts of interest.

 All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes