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ASSOCIATION BETWEEN MUTATIONS IN PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER AND P. FALCIPARUM MULTIDRUG RESISTANCE 1 GENES AND IN VIVO AMODIAQUINE RESISTANCE IN P. FALCIPARUM MALARIA–INFECTED CHILDREN IN NIGERIA

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  • 1 Malaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria; Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts; Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Springs, Maryland; Special Program for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58–36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

INTRODUCTION

The emergence and spread of parasites resistant to antimalarial drugs continues to be a major public health problem in the management of Plasmodium falciparum infections in many malaria-endemic countries. Resistance to chloroquine (CQ), the most widely used and affordable antimalarial drug, has contributed to increased mortality and morbidity caused by P. falciparum infections.1,2 Increasing CQ and sulfadoxine-pyrimethamine resistance in P. falciparum has led to renewed search for alternative effective drugs and efforts to counter the problems of resistance to these two major antimalarial drugs. Many malaria-endemic countries in Africa are currently facing the crucial issue of switching drug regimens. There is an increasing acceptance that the ideal approach to antimalarial treatment is the use of combination of two or more drugs, rather than a single antimalarial drug, preferably with artemisinin derivative as one of the drugs.3 Amodiaquine (AQ) in combination with artesunate (AS) has been introduced as first-line treatment of malaria to replace CQ in Nigeria and other malaria-endemic countries of Africa.4 Although the role of AS in this combination is to prevent the development of AQ resistance, parasites may quickly develop resistance to AQ in areas where extensive CQ resistance has been documented. In addition, little is known about the mechanism or epidemiology of AQ resistance.5 Cross-resistance between CQ and AQ has been reported both in vitro6,7 and in vivo.812 Furthermore, increasing use of AQ will increase emergence of P. falciparum strains with reduced sensitivity to this drug. Therefore, a tool to monitor the development and spread of resistance to AQ is greatly needed.

Despite reports of cross-resistance between CQ and AQ, the molecular mechanisms of AQ resistance and the role of mutations in the P. falciparum chloroquine resistance transporter (pfcrt) or P. falciparum multidrug resistance 1 (pfmdr1) genes remain unclear. The similarity in the chemical structures of CQ and AQ13 and their likely common mode of action13,14 is suggestive that molecular markers of CQ resistance may be useful for detection of parasites resistant to AQ. Two parasite proteins associated with the lysosomal membrane, Pgh1 (the protein product of the pfmdr1 gene) and PfCRT (the protein product of the pfcrt gene), are thought to be important to the process of CQ resistance. Point mutations in two P. falciparum genes encoding these proteins have been associated with in vitro CQ resistance in laboratory lines or field isolates,1522 and have been used as molecular markers of CQ resistance in vivo in many malaria-endemic countries.2334 In this study, the association between pfcrtT76 and pfmdr1Y86 alleles and AQ treatment outcome in children with acute uncomplicated P. falciparum malaria was investigated in Ibadan in southwestern Nigeria. We showed that the combination of mutant pfcrtT76 and pfmdr1Y86 alleles was associated with in vivo AQ resistance and that this association was age dependent. In addition, analysis of post-treatment samples obtained from patients who failed therapy showed that AQ selects parasites with pfcrtT76 and pfmdr1 alleles. Furthermore, we explored the possibility of using these markers as tools for monitoring the emergence and spread of resistance to AQ in countries that have introduced AQ in combination with other antimalarials as first-line treatment of malaria.

METHODS

Study area.

The study was carried out at the Malaria Research Laboratories, College of Medicine, University of Ibadan, Nigeria, from April to September 2005. Malaria in Ibadan is hyperendemic and transmission occurs year round, but is more intense from April to October during the rainy season.

The protocol for the study was reviewed and approved by the Joint University of Ibadan/University College Hospital Institutional Review Committee at the University of Ibadan, Nigeria and the Harvard School of Public Health Human Subject Committee. Documented informed consent was obtained from parents and guardians.

Patient treatment and follow-up.

Children were eligible to join the study if they were ≤ 12 years of age, had symptoms of acute uncomplicated malaria, with pure P. falciparum parasitemia between 2,000 and 500,000 asexual forms/μL of blood, a temperature ≥ 37.5°C or recent history of fever, absence of other concomitant illness, no history of antimalarial use in the two weeks preceding presentation, negative urine test results for antimalarial drugs (Dill-Glazko and Lignin), and written informed consent given by parents or guardians. Patients with severe malaria,35 severe malnutrition, serious underlying diseases (renal, cardiac, or hepatic), and known allergy to study drugs were excluded from the study. The disease history was recorded by asking patients or their parents when the present symptomatic period had started, and was followed by a full physical examination.

Enrolled children were treated with standard regiment of AQ (30 mg/kg of body weight over a three-day period). The drug was given orally in the clinic and all patients were observed for at least 1 hour after drug administration to ensure the drug was not vomited. Children who vomited the medication were excluded from the study. If necessary, children were provided with antipyretics (paracetamol tablets, 10–15 mg/kg every 8 hours for 24–48 hours). A physician controlled drug administration. Follow-up with clinical and parasitologic evaluation was done daily for seven days (days 1–7) and then on days 14, 21, and 28. Thick and thin blood films prepared from a finger prick were stained with Giemsa and examined by light microscopy under an oil-immersion objective at 1,000× magnification by two independent assessors. Parasitemia (asexual or sexual) in thick films was estimated by counting asexual or sexual parasites relative to 1,000 leukocytes. From this figure, the parasite density was calculated assuming a leukocyte count of 8,000/μL of blood. Two drops of blood was also blotted onto 3MM filter paper (Whatman International Ltd., Maidstone, United Kingdom) for extraction and analysis of parasite DNA.

Classification of responses to treatment was done according to World Health Organization criteria.36 The cure rate on day 28 of follow-up was defined as the percentage of children who remained free of parasitemia. Treatment failures rates were corrected by merozoite surface protein 2 (msp-2) genotyping of parasites at enrollment and recrudescence of infections.33,37 Children who failed treatment (within 14 days) with AQ were re-treated with artemether (9.6 mg/kg over a five-day period and were regarded as treatment failures). Children were re-treated whenever they became symptomatic (usually between 14 and 21 days after initial enrollment).

Extraction of DNA from samples collected on filter paper.

Parasite genomic DNA was extracted from blood samples collected on filter paper using the Chelex extraction method as described by Plowe and others.38 Part of the DNA extracted from each sample was used immediately for polymerase chain reaction (PCR) and the rest was stored at −20°C.

Amplification of P. falciparum genes and characterization of parasites population in infected patients.

The regions of the pfcrt and pfmdr-1 genes surrounding the polymorphisms of interest (in the smear-positive samples collected pre-treatment and post-treatment) were amplified by nested PCR and then subjected to restriction fragment length polymorphism (RFLP) analysis as previously described.32,33 In addition, each P. falciparum infection in this study was characterized on the basis of msp-2 polymorphism performed on paired pre-treatment and post-treatment parasites samples obtained from patients.33,37 This genetic marker was chosen because a recent report33 from the same study site has demonstrated that msp-2 is the best and most reliable marker to evaluate diversity and complexity of P. falciparum infections in both pre-treatment and post-treatment isolates because it showed more clones than other markers (msp-1 or glutamate-rich protein). Thus, msp-2 analyses of paired pre-treatment and post-treatment parasites were used to distinguish true treatment failures from new infections. The complexity of infection was calculated as the average number of distinct fragments of FC27 and IC1/3D7 per PCR-positive sample.33,37

Statistical analysis.

For a best assessment of genetically determined parasite phenotypes, only parasitologic treatment failures were used to define AQ resistance. For analysis purposes, each isolate was coded based on the presence or absence of a resistance-associated allele. For example, infections with mixed wild-type/mutant alleles of pfcrt or pfmdr1 were treated as mutants. Data were analyzed using the statistical programs SPSS for Windows version 10.01 (SPSS, Chicago, IL) and Epi-Info version 6.4 (Centers for Disease Control and Prevention, Atlanta, GA). For univariate analysis, frequencies were compared by calculating chi-square values with Yates’ correction and Fisher’s exact tests. Normally distributed, continuous data were compared by Student’s t-tests and analysis of variance. Paired samples were compared using the paired t-test. Data not conforming to a normal distribution were compared by the Mann-Whitney U test (or Wilcoxon ranked sum test) and the Kruskal-Wallis test. Multiple logistic regression analysis was performed to assess the weight of all variables on treatment outcome and clearance of resistant parasites. All tests of significance were two-tailed. P values < 0.05 indicated statistical significance.

RESULTS

Patient treatment outcomes.

A total of 106 children with acute uncomplicated malaria were enrolled into the study and treated with AQ. Characteristics and demographic data of the children are shown in Table 1. Of these children, 101 (95%) successfully completed the 28-day follow-up. Data from the remaining 5% of the patients who did not complete the 28-day follow-up were excluded from analysis. Treatment failures were confirmed by msp-2 genotyping. Overall, 87% (88 of 101) of the patients were cured by AQ, and 13% (13 of 101) of the patients failed AQ treatment. Resistance levels in all children who failed treatment with AQ were RI. Age-stratified analysis of patients who failed treatment showed that 54% (7 of 13) of these patients were ≤ 5 years of age, and 46% (6 of 13) were > 5 years of age. Age (P = 0.240) or parasite density (P = 0.999) at enrollment were not significantly associated with AQ treatment failure.

Determination of P. falciparum clonal profiles in malaria-infected patients isolates.

Matched sample pairs collected before and after treatment from all 13 patients who failed AQ treatment were successfully analyzed at the msp-2 locus. Presence of different allelic families of msp-2 was often found in parasite DNA derived from a single patient, indicating a polyclonal infection. The estimated mean ± SD number of genetically distinct parasite populations as determined with msp-2 in pre-treatment and post-treatment isolates from these 13 patients was 4.38 ± 0.66 and 2.23 ± 1.2, respectively. There was a significant reduction (P = 0.028) in the number of msp-2 alleles in post-treatment isolates compared with pre-treatment isolates when infections recrudesced. Eighty-five percent (11 of 13) paired PCR fragments were identical at both FC27 and IC1 allelic families of msp-2, indicating genuine recrudescent infections after treatment with AQ. Infections in the remaining two (15%) patients showed parasites similar to pre-treatment isolates and the presence of new parasite populations with different genotypes. However, we considered these infections as resulting from genuine treatment failures.

Prevalence of pfmdr1 and pfcrt mutations in patients isolates of P. falciparum.

Molecular assays were performed on all isolates collected from patients prior to treatment. The presence of pfcrtT76 and pfmdr1Y86 mutations known to be involved in resistance to CQ treatment in west Africa was evaluated. Mutation analysis was successful in all 101 of the isolates collected from the patients. Results for pfcrt and pfmdr1 mutations evaluated are shown in Table 2. The prevalence of mutant pfcrtT76 (62%) allele was higher than that of the mutant pfmdr1Y86 (29%) allele. However, mixed pfmdr1Y86 + pfmdr1N86 (28%) or pfcrtT76 + pfcrtK76 (16%) alleles were common in many samples. The prevalence of both mutant pfcrtT76 and pfmdr1Y86 alleles (46%) was also evaluated in samples collected before drug treatment (Table 2).

Association between pfcrt and pfmdr1 point mutations and failure to treatment with AQ.

To test our hypothesis whether allelic variations in P. falciparum pfcrt or pfmdr1 are associated with outcome of patients treated with AQ, the presence of single pfcrtT76 or pfmdr1Y86 or the combination of these two mutations (pfcrtT76 + pfmdr1Y86) in samples collected prior to treatment with AQ were examined for their association with patients treatment outcome. Each isolate was coded based on the presence or absence of a resistance-associated allele. For example, infections with mixed wild-type/mutant alleles were treated as mutant. The mutant pfcrtT76 (P = 0.065) allele was not independently associated with AQ treatment failure (Table 3). The mutant pfmdr1Y86 allele was weakly associated (odds ratio [OR] = 5.02, 95% confidence interval [CI] = 1.051–23.98, P = 0.036) with AQ treatment failure. However, when the relationship between the combination pfcrtT76 + pfmdr1Y86 alleles and AQ treatment failure was investigated, a stronger association was observed between children infected with parasites harboring the combination of mutant pfcrtT76 and pfmdr1Y86 (OR = 7.58, 95% CI = 1.58–36.25, P = 0.006) (Table 3). To further examine the role of these mutations on AQ treatment failure, we compare the prevalence of mutations in baseline samples (samples obtained from all patients) and post-treatment samples obtained from patients who failed treatment. The analysis showed that the pfcrtT76 allele (P = 0.067) was not significantly selected by AQ treatment, while the mutant pfmdr1Y862 = 14.44, P = 0.00024) or the combination of mutant pfcrtT76 and pfmdr1Y862 = 10.08, P = 0.0015) were strongly selected by the drug (Table 4).

Impact of age and pfcrt/pfmdr1 mutations on patient treatment outcome.

The effect of age on the association between pfcrt and pfmdr1 mutations and treatment outcome was evaluated in patient samples from two different age groups (≤ 5 years of age and > 5 years of age). Only 9 (9%) of 101 samples obtained at enrollment prior to treatment had no mutation in both genes, and all patients with these infections were cured with AQ regardless of the age category. The associations between the presence of both mutant pfmdr1Y86 and pfcrtT76 alleles and treatment failures was significant (OR = 10, 95% CI = 1.07–93.43, P = 0.035) only in children ≤ 5 years of age and not in older children (> 5 years of age) (OR = 2.26, 95% CI = 1.33–3.92, P = 0.405).

Pfcrt/pfmdr1 mutants and parasites clearance.

The role of some patient characteristics on the ability to clear the infections with pfcrt/pfmdr1 mutant parasites was evaluated by analyzing the potential association between parasites clearance rates, age, parasites density or fever (body temperature ≥ 37.5°C) at enrollment as described previously by Djimde and others.39 Univariate analysis showed that the clearance of pfcrtT76 and pfmdr1Y86 mutants parasites by patients was significantly associated with age (P = 0.0017), and fever at enrollment (χ2 = 7.36, P = 0.0066) (Table 5). There was no association between pfcrt/pfmdr1 mutant parasites clearance and parasite density (P = 0.7). However, when logistic regression analysis was performed to confirm the weight of univariate analysis results (age and fever at enrollment), only age was independently associated with the clearance of the resistant phenotype (P = 0.012). Children ≤ 5 years of age showed the lowest rate (55%, n = 11) of clearance of resistant parasites compared with much older children who showed a higher rate (84%, n = 26) of clearance of pfcrt/pfmdr1 mutant parasites.

DISCUSSION

Overall, the current study shows an increase in resistance to AQ from 4.8%11 to 13% in Nigeria between 2001 and 2005. Data from the study also showed a selection of mutant pfcrtT76 and pfmdr1Y86 alleles by AQ in children who failed treatment. More importantly, the present study demonstrated the association between the presence of both mutant pfcrt and pfmdr1 in combination and in vivo AQ resistance and the important role of age (the best surrogate marker for immunity) in clearing AQ-resistant parasites in an area of intense malaria transmission in west Africa.

The data from this study show an increase in rate of resistance to AQ in the study area. This increase might have been exacerbated by the extensive use of CQ in Nigeria. Cross-resistance between CQ and AQ has been observed in the study area10 and other disease-endemic areas.40 The increased in vivo AQ resistance in Nigeria observed in this study is of great concern because this drug is currently being used in this country in combination with AS for the treatment of acute uncomplicated malaria. A study in the Gambia41 has shown using reverse transcriptase-PCR that low density sub-populations of asexual parasites can escape short-acting drugs such as AS. Resistant parasites then likely to recrudesce under the selective force of the second drug in the combination and be transmitted to mosquitoes.41 Therefore, we cannot rule out the possibility of increasing selection of AQ-resistant parasites with the increasing use of AQ in combination with artesunate in the future in Nigeria.

Molecular methods that detect genetic markers of drug resistance are potentially powerful tools for tracking drug-resistant malaria. In this study, the combination of pfcrt and pfmdr1 mutations in isolates obtained from children prior to treatment was associated with in vivo AQ resistance during a 28-day clinical efficacy trial. Mutant pfcrtT76 and pfmdr1Y86 alleles were observed in 62% and 29% of the samples, respectively. The mutant pfcrtT76 allele was not a useful predictor of clinical outcome, and the mutant pfmdr1Y86 allele weakly predicted patients’ clinical outcome. The data from this study are different from a previous study in Sudan,42 which found that the mutant pfcrtT76 allele is associated with AQ treatment failure. This may be explained by differences in the epidemiology of CQ resistance between the two study sites and the high prevalence of the pfcrtT76 allele observed in Ibadan in southwestern Nigeria.

The high prevalence of the mutant pfcrtT76 allele (62%) observed in Ibadan, Nigeria confirms recent reports of the high prevalence rate of this allele in parasites obtained from same study site,32,33 and is also consistent with rates ranging from 60% to 100% reported in other malaria-endemic regions.25,2931,34 However, analysis of baseline samples (samples collected from all patients at enrollment) and post-treatment samples from patients who failed AQ treatment showed the selection of parasites with mutant pfcrtT76 or pfmdr1Y86 or both mutant alleles by the drug (Table 4), although this selection was significant only for parasites with the mutant pfmdr1Y86 allele or both mutant pfcrtT76 and pfmdr1Y86 alleles. This selection process was further confirmed by msp-2 analysis, which showed a reduction in the average number of distinct clones of P. falciparum per infection from 4.38 at enrollment to 2.23 at recrudescence.

The non-significant selection of the mutant pfcrtT76 by AQ may be due to the high prevalence of this allele in the P. falciparum population from Ibadan, Nigeria.32 Selection of pfmdr1Y86 by AQ has also been reported previously in the Gambia19 and Kenya.43 Although the importance of point mutations in pfcrt in producing CQ resistance is beyond dispute,4447 recent transfection studies of pfcrt have shown that isolates expressing the mutant pfcrtT76 allele retain sensitivity to AQ while showing a reduced susceptibility to monodesethyl AQ, the active metabolite of AQ.44 Therefore, it is possible that during treatment with AQ, monodesethyl AQ may be the driving force behind the selection of the mutant pfmdr1Y86 or pfcrtT76 alleles or the combination of these mutant alleles as observed in post-treatment samples of patients who failed treatment. The selection of the mutant pfcrtT76 and pfmdr1Y86 alleles indicates the primary involvement of these two genes in the mediation of AQ resistance. Most importantly, the absence of the wild-type pfcrtK76 allele in post-treatment samples of patients who failed treatment (Table 4) may indicate the critical role of the mutant pfcrtT76 allele in AQ resistance. Thus, similar to CQ resistance, AQ resistance in P. falciparum may depend primarily on mutation(s) in pfcrt and additional mutations in pfmdr1 or other Plasmodium genes may also have significant roles in increasing resistance to the drug.

The combination of pfcrtT76 and pfmdr1Y86 mutations was associated with AQ treatment failure. These two alleles have been shown to be in linkage disequilibrium in CQ-resistant isolates of P. falciparum from The Gambia48 and Nigeria49 (Happi CT and others, unpublished data). The similarity in the chemical structures of CQ and AQ13 and their likely common mode of action13,14 suggests that the molecular basis of resistance to these two drugs may be similar. These previous findings may explain the strong association (P = 0.006) between the combination of pfcrtT76 and pfmdr1Y86 mutant alleles and in vivo AQ failure observed in this study, although, age-stratified analysis of the effect of these two mutations on treatment outcome did not show a significant association with treatment failure in children more than five years of age. One possible explanation for this finding is the immunopotentiation of AQ efficacy by the hosts. Previous studies have shown that in areas of moderate or high malaria transmission, drugs with suboptimal efficacy could cure infections in patients (>5 years of age) who have developed a degree of antiparasitic immunity to malaria (due to repeated exposure to various circulating drug-sensitive and drug-resistant strains of parasites).36,50,51

The ability of some children in this study to clear infections with parasites with both pfcrtT76 and pfmdrY86 mutations was strongly associated with age (> 5 years) and fever at enrollment. The association between fever at enrollment and clearance of drug-resistant parasites is most likely related to an appropriate cytokine response (i.e., tumor necrosis factor-α, interferon-γ) in the children clearing peripheral parasitemia. However, the synergy between the appropriate fever response of the host and drug treatment is unclear in light of this finding. The data from this study further show that children less than five years of age are highly vulnerable to malaria and cannot clear their parasites as efficiently as other age groups (Table 5). These data are similar to those in a previous report from Mali.39

Overall, the findings from this study have significant bearings and implications on drug discovery and public health in Nigeria where AQ is combined with other antimalarial drugs (including artemisinin derivatives) for treatment of acute uncomplicated malaria. First, the evidence of selection of mutant pfcrtT76 and pfmdr1Y86 alleles by AQ may provide some insight in the previously observed cross-resistance between CQ and AQ in vivo.11,40 Second, mutant pfcrtT76 and pfmdr1Y86 alleles currently used as molecular markers of CQ resistance can also be useful for monitoring the spread of AQ resistance in areas of low resistance to the drug such as west Africa. Third, clearance of the AQ-resistant phenotype by children less than five years of age suggests that for countries that have not yet changed their antimalarial drug policy, changes aimed at switching first-line treatment to drug combinations could be initially targeted to children less than five years of age, especially when the cost of these combinations is an obstacle to implementing the policy.

Ultimately, there is a need to validate mutant pfcrtT76 and pfmdr1Y86 alleles as markers for AQ resistance as proposed in this study in other malaria-endemic areas. Such an approach is necessary because it may harness the efforts aimed at the management and control of drug-resistant malaria.

Table 1

Enrollment clinical data and therapeutic responses of patients with acute uncomplicated Plasmodium falciparum malaria treated with amodiaquine

Treatment with amodiaquine
No. enrolled101
Age (years), mean ± SD6 ± 3.00
Range (years)1–12
Sex (%)
    Males54 (53.5)
    Females47 (46.5)
Male:female ratio1.15:1
Body temperature (°C), mean ± SD38.14 ± 1.16
Range (°C)36.10–40.90
Geometric mean parasite density (per μL of blood)23,033
    Range2,070–180,390
Therapeutic responses
    Fever clearance time (days), mean ± SD1.21 ± 0.67
    Range (days)1–5
    Parasite clearance time (days), mean ± SD2.57 ± 0.88
    Range (days)1–5
    Responses by day 28, no. (%)
        Cured88 (87%)
        Resistant-RI13 (13%)
        Resistant-RII0 (0%)
        Resistant-RIII0 (0%)
Table 2

Baseline frequency and prevalence of pfmdr1 at codon 86 and pfcrt alleles at codon 76 in isolates collected from Nigerian children (n = 101) at enrollment before treatment with the standard dosage of amodiaquine*

Gene and allelesFrequencyPrevalence (%)
* pfmdr1 = P. falciparum multidrug-resistance 1; pfcrt = P. falciparum chloroquine resistance transporter; K = lysine; T = threonine; N = asparagine; Y = tyrosine.
pfcrt at codon 76 (n = 101)
    Wild-type K76 (AAA)2222
    Mutant T76 (ACA)6362
    K76 and T761616
pfmdr1 at codon 86 (n = 101)
    Wild-type N86 (AAT)4443
    Mutant Y86 (TAT)2829
    N86 and Y862928
Table 3

Association between pfcrt/pfmdr1 mutations in Plasmodium falciparum isolates colllected at enrollment and amodiaquine treatment failure*

Treatment outcome (%)
AllelesFailedCuredOR (95% CI)P
* Treatment failure was based on the World Health Organization 28-day drug efficacy test and monitoring36 and corrected by merozoite surface protein 2 genotyping of infections.33,37 All infections with mixed wild-type/mutant alleles of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene or P. falciparum multidrug resistance 1 (pfmdr1) gene were treated as mutants. OR = odds ratio; CI = confidence interval.
† Statistically significant.
pfcrt
    T7613/13 (100)67/88 (76)1.31 (1.16–1.47)0.065
    K760/13 (0)21/88 (24)
pfmdr1
    Y8611/13 (85)46/88 (52)5.022 (1.051–23.98)0.036†
    N862/13 (15)42/88 (48)
pfmdr1 and pfcrt
    pfmdr1Y86 + pfcrtT7611/13 (85)37/88 (42)7.58(1.58–36.25)0.006†
    pfmdr1N86 + pfcrtT762/13 (15)51/88 (58)
Table 4

Selection of mutant pfcrtT 76 and pfmdr1Y86 alleles in Nigerian children by amodiaquine treatment: prevalence of point mutations in pfmdr1/pfcrt genes among baseline infections and at recrudescence of infections in children after amodiaquine treatment*

Prevalence of point mutations in patient samples (n)
AllelesBaselineRecrudescenceχ2P
* For definitions of abbreviations, see Table 2.
† Statistically significant.
pfmdr1 codon 86
    Wild-type N8643% (44/101)8% (1/13)5.910.013†
    Mutant Y8629% (29/101)84% (11/13)13.440.000246†
    Mixed N86 + Y8628% (28/101)8% (1/13)1.490.221
pfcrt codon 76
    Wild-type K7622% (22/101)0% (0/13)2.250.133
    Mutant T7662% (63/101)92% (12/13)3.310.0671
    Mixed K76 + T7616% (16/101)8% (1/13)0.130.716
pfmdr1 codon 86 + pfcrt codon 76
    N86 + K7612% (12/101)0% (0/13)0.700.404
    Y86 + T7646% (47/101)92% (12/13)10.080.0015†
    Mixed (N86 + T76/Y86 + K76)42% (41/101)8% (1/13)4.280.038†
Table 5

Association between patients characteristics and clearance of amodiaquine-resistant Plasmodium falciparum in children from Nigeria*

Patients clearing double-mutant parasites (n)Patients not clearing double-mutant parasites (n)P
* Patients harbored mutant alleles of both the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multidrug resistance 1 (pfmdr1) gene (pfcrtT76 + pfmdr1Y86).
† Statistically significant by Fisher’s exact test.
Mean ± SD, years6.42 ± 2.57 (37)3.90 ± 2.57 (11)0.0017†
Fever89.2% (33)45.4% (5)0.0066†
Mean log parasite density10.25 ± 0.939.96 ± 1.180.4

*

Address correspondence to C. T. Happi, Malaria Research Laboratories IMRAT, College of Medicine, University of Ibadan, Nigeria. E-mail: christianhappi@hotmail.com or chappi@hsph.harvard.edu

Authors’ addresses: C. T. Happi, Malaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria and Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115. G. O. Gbotosho, O. A. Folarin, O. M. Bolaji, and A. Sowunmi, Malaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria. D. E. Kyle and W. Milhous, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Springs, MD 20910. D. F. Wirth, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115. A. M. J. Oduola, Special Program for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland.

Acknowledgments: We thank all patients and their parents or guardians for volunteering to participate in the study, and the Malaria Research and Reference Reagent Resource Centre for providing all genomic DNA used as controls for the PCR and RFLP experiments. We also thank Dr. Dan Milner (Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA) for helpful comments on the manuscript.

Financial support: This study was supported by a grant from the Fogarty International Center, Multilateral Initiative for Malaria in Africa/TDR, project ID A20239, UNICEF/UNDP/World Bank/WHO/TDR grant project ID A50337, Harvard Malaria Initiative, and International Atomic Energy Agency (IAEA) project RAF/0625. C. T. Happi is supported by a Fogarty International Research Collaboration Award (FIRCA) no. NIH RO3TW006298-01A1, the IAEA project RAF/0625, and the UNICEF/UNDP/World Bank/WHO/TDR/PAG/South-South Initiative project ID A50337.

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