1 Secretaría Departamental de Salud del Valle, Cali, Colombia.
2 Escuela de Salud Pública, Universidad del Valle, Cali, Colombia.
3 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD.
4 Departamento de Microbiología, Universidad del Valle, Cali, Colombia.
5 Fundación para la Educacion Superior, Cali, Colombia.
6 Instituto de Inmunología, Universidad del Valle, Cali, Colombia.
7 Malaria Section, Center for Vaccine Development, University of Maryland, Baltimore, MD.
Received for publication September 5, 2001; accepted for publication March 15, 2002.
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ABSTRACT |
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dihydropteroate synthase; drug resistance; malaria; Plasmodium falciparum; pyrimethamine; sulfadoxine
Abbreviations: Abbreviations: DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthase; PCT, parasite clearance time; SP, sulfadoxine-pyrimethamine; WHO, World Health Organization.
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INTRODUCTION |
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The molecular mechanisms of antifolate resistance have been clearly defined in vitro. Point mutations in the P. falciparum genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) confer resistance to pyrimethamine and sulfadoxine, respectively (48). These resistance-conferring mutations occur in a stepwise, sequential fashion, with higher levels of in vitro resistance occurring in the presence of multiple mutations. However, clinical SP failure is an outcome of a complex interplay of host, parasite, and drug, and identification of specific sets of mutations predictive of clinical resistance has been difficult (1, 9). An ecologic analysis found increasing prevalence of DHFR and DHPS mutations in four countries with increasing levels of SP resistance (3), and various sets of DHFR and DHPS mutations have been associated with SP failure in settings with relatively high levels of SP therapeutic failure (10, 11). Recently, a set of five DHFR mutations and two DHPS mutations was shown to be strongly associated with SP failure in Malawi (12), and new models that account for host factors contributing to parasite clearance may permit use of these mutations as molecular markers for drug resistance surveillance (13). However, the effects of parasite mutations on treatment outcomes in settings of low antifolate resistance are less well understood, and the factors involved in the vector-host transmission of drug resistance remain imprecisely defined.
In this study of 120 subjects from the Pacific coast of Colombia, we assessed the association between the occurrence of mutations in P. falciparum DHFR and DHPS genes with SP treatment response and also with subsequent development of gametocytemia (carriage of the sexual forms of the parasite required for transmission by the mosquito). This study sought to contribute to an understanding of the molecular basis of treatment response to SP and to explore the impact of DHFR and DHPS mutations on transmission potential.
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MATERIALS AND METHODS |
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Standard SP treatment was administered under direct observation as a single dose of 1.25 mg pyrimethamine and 25 mg sulfadoxine per kg of body weight (maximum dose, 75 mg pyrimethamine and 1,500 mg sulfadoxine). Fever was treated with acetaminophen. Subjects who developed SP treatment failure were hospitalized, treated with intravenous quinine and/or amodiaquine, and followed until full recovery. All subjects were examined on posttreatment days 1, 2, 3, 7, 14, and 21 for determination of parasitemia and a clinical assessment including measurement of body temperature. Persons whose infections had not cleared on day 3 were seen the following day to monitor treatment response status. Patients were encouraged to come to the clinic at any time for any medical complaint.
Exposures of interest
The primary exposures of interest in this study were mutations in the parasite genes encoding DHFR and DHPS. Other exposures included drug concentrations, nutritional status, and potential confounders.
Assessment of parasite mutations
Finger-prick blood was collected on filter paper strips (Whatman, Hillsboro, Oregon), air dried, stored in separate envelopes, and transported at room temperature. After the blood was stored for several weeks at 4°C, DNA was extracted from pieces of blood-impregnated filter paper approximately 3 mm2 by using a methanol fixation-heat extraction method (16). Polymerase chain reaction protocols have been described in detail elsewhere (3) and are available on the Internet at http:// medschool.umaryland.edu/CVD/plowe.html. Briefly, a nested, allele-specific polymerase chain reaction was performed for the analysis of each codon of interest. A pilot study conducted at the study site did not detect infections with mutations at DHFR codons 50 or 59 or at DHPS codon 581 (results not shown). Our analysis was therefore limited to the DHFR mutations at codons 108 (serine to asparagine), 51 (asparagine to isoleucine), and 164 (isoleucine to leucine) and the DHPS mutations at codons 437 (alanine to glycine) and 540 (lysine to glutamate).
Assessment of drug concentrations
Plasma was frozen in duplicate for determination of pyrimethamine and sul-fadoxine concentrations 24 hours after treatment. Blood drug levels were determined using high performance liquid chromatography.
Assessment of nutritional status
We determined body mass index (weight (kg)/height (m2)) and percentiles of middle arm circumference and calculated z scores of the ratios weight:height and height:age by using the Centers for Disease Control and Prevention anthropometric software program Epi-nut (Centers for Disease Control and Prevention, Atlanta, Georgia). Standard criteria for age and gender limits of hematocrit and iron (17) were used to determine the presence of anemia.
Other potential confounders
At enrollment, we recorded age, gender, place of residence, report of microscopically diagnosed malaria during the preceding year, time from onset of illness to seeking treatment, and antimalarial drug intake prior to enrollment.
Outcome measures
Treatment outcomes
We used two measures of treatment response: incidence of treatment failure and parasite clearance time (PCT). Treatment outcome was classified as adequate clinical response or early or late treatment failure, using standard WHO criteria (14). PCT was defined as the time from starting antimalarial treatment until parasites were undetectable in the peripheral blood film. An experienced laboratory technician measured asexual parasitemia (asexual parasites/µl blood) by reading 200 high-power fields on Giemsa-stained thick blood films.
Transmission potential
We evaluated the potential for transmission by measuring gametocytemia on enrollment and at scheduled visits following the same technique used for asexual parasitemia.
Statistical methods
We calculated the proportion of malaria infections with mutations of interest present at the time of enrollment and estimated 95 percent binomial confidence intervals for prevalence of mutations in the study area. Since PCT was assessed at 24-hour intervals, a survival analysis for discrete time data (days to parasite clearance after treatment) was carried out using log-rank tests to compare PCT in persisting infections with or without each mutation of interest. This analysis is equivalent to the exact Mantel-Haenszel test or the score statistic of a Cox regression using exact likelihood methods (18). Extension of the methods for the analysis of several factors results in a semiparametric regression to estimate relative hazards and their 95 percent confidence intervals (18). The exact partial likelihood method was used to treat ties. With this method, the likelihood is changed to reflect the discreteness of time measures, and the conditional probability that the observed events are the ones that occurred in the risk pool given the observed number of events is calculated (18).
Subjects were classified by categories of putative determinants of treatment response (e.g., anemic or not, quartiles of initial parasitemia). To evaluate the role of potential determinants of treatment response as confounders, we characterized their relation with both the exposure (mutations) and the outcome (PCT). Based on results of the univariate analysis and previous knowledge of determinants of treatment response, a multiple Cox regression model was used to obtain the association of mutations and PCT adjusted by other confounders (18).
We used two descriptors for the distribution of gametocytemia: 1) the proportion of persons with detectable gametocytes at each scheduled visit (percentage greater than zero or discrete component), and 2) mean gametocytemias (in the log10 scale), excluding those with no gametocytes (mean for those that are greater than zero or continuous component). Chi-square tests for trend were conducted to evaluate the dose-response relation between mutations and gametocytemia by each day of follow-up after treatment. Evaluation of significant differences for means of gametocytemias was carried out by linear regression.
Because it was possible that an effect of mutations on gametocytemia could be mediated through PCT, we mea-sured the extent of an indirect effect of mutations on gametocytemia via PCT by comparing the logistic regression analysis to estimate the odds ratio of developing gametocytemia according to categories of mutant parasites with that of a model including adjustment for PCT.
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RESULTS |
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Table 1 summarizes the prevalence of DHFR and DHPS mutations. Most of the samples (80 percent) that could not be amplified were more than 10 months old and/or had parasitemias below 5,000 parasites/µl. Mutations at DHFR codon 164 and DHPS codon 540 were not found in the analysis of the first 52 and 70 samples, respectively, and assays for these mutations were discontinued thereafter.
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Determinants of treatment response
Four subjects developed treatment failure (4/120 = 3.3 percent), with one early and three late treatment failures. Figure 1 depicts the trajectories of log10 parasitemias for all subjects, including the four treatment failures. To avoid ties in the graphic depiction, we randomly added a small quantity (between 0 and 0.5) to each value of parasitemia. The cumulative PCT distribution for the 120 study subjects was as follows: 24.2, 83.4, 95.8, and 96.7 percent cleared parasites by days 1, 2, 3, and 4, respectively. Subjects with a PCT of longer than 3 days (n = 5) included four persons with treatment failure and one subject with adequate clinical response that cleared parasitemia on day 4.
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The presence of mutations at both DHFR codons was more strongly associated with longer PCT (relative hazard for clearance = 0.24, p = 0.019) than that of the mutation at codon 108 alone (relative hazard = 0.45, p = 0.188) (table 2). Since the categorical analysis suggested a dose response, we carried out an analysis with a single variable, taking values 0, 1, and 2 for no mutations, 108 alone, and both the 108 and 51 mutations, respectively. With this continuous variable, the hazard ratio was 0.51 (p = 0.012), showing a significant trend of PCT being halved as an additional mutation is involved in the malaria infection.
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Multivariate analysis of treatment response
We evaluated confounding effects among the potential determinants of PCT. Because our primary interest was to evaluate parasite mutations as potential determinants of treatment response, we built a model selecting variables from factors that appeared to be important in the univariate analysis (pyrimethamine concentration, parasitemia on day 0, and low iron plasma concentration) or that had an important biologic association (age, gender, malaria episodes in the previous year, time to diagnosis, and chloroquine intake prior to therapy). We fitted a multiple Cox regression model for the variables that remained associated at p < 0.15 (table 3). Inferences regarding the effect of mutations drawn from the univariate analysis remained after adjustment by levels of parasitemia and drug concentration.
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Multivariate analysis for gametocytemia
Using multiple logistic regression, we estimated the odds ratio for the association between mutations (as predictor variables) and the occurrence of gametocytemia, unadjusted and adjusted for duration of PCT (table 4). Because odds ratios of mutations tend to be closer to one when adjusted by duration of PCT, the results in table 4 indicate that part of the association of mutations and gametocytemia is indirect and can be explained by longer PCT. However, particularly at day 14, mutations at DHFR genes remained significantly associated with gametocytemia even after adjustment by duration of PCT. Results remained unchanged after further adjustment by asexual parasitemia at baseline.
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DISCUSSION |
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Clearance of parasites took more than 1 day in 75.8 percent of the subjects and more than 2 days in 17.6 percent of the subjects. Our results are similar to those recently reported in a study conducted in The Gambia in which 81 percent of the children treated with SP were still parasitemic on day 1 and 19.2 percent were parasitemic on day 2 (19).
We evaluated five mutations in P. falciparum DHFR and DHPS that have been reported to be associated with SP resistance. As reported in studies from other countries with low incidence of treatment failure (3), only three of these mutations were found in our study: the DHFR mutations at codons 108 and 51 and the DHPS mutation at codon 437. The mutation at DHFR codon 164, which was strongly associated with treatment failure in studies made in Bolivia and Peru (3, 20), was not found in this study.
We showed that falciparum infections clear more slowly if they carry parasites with DHFR mutations. Furthermore, the presence of both DHFR mutations at codons 108 and 51 was more strongly associated with PCT than that of the mutation at codon 108 alone (relative hazard = 0.24 and 0.45, respectively), which supports the enhancing effect of multiple mutations in the in vivo response as has been described (3, 11, 12, 20). We did not find an association of PCT with the DHPS 437 mutation, although this mutation was always found in infections with doubly mutant DHFR, precluding an independent analysis of its contribution to PCT. This DHPS 437 mutation is common in epidemiologic surveys even in areas with sparing use of antifolates (3) and, like the DHFR serine to asparagine 108 mutation, while it may be a necessary first step toward a more fully resistant infection, by itself it is only a minor contributor to antifolate resistance (21). The impact on PCT of multiply mutant DHPS, which was not identified in this study, remains to be assessed.
We further evaluated the consistency of the association of DHFR mutations with PCT by considering potential confounding effects. In particular, parasitemia level at admission was significantly associated with PCT in our study and was also strongly associated with PCT in a study of predictors of mefloquine treatment in Thailand (22). Patients with higher parasitemia will have longer PCT, even if the infection is fully sensitive, because there are more parasites in the body to be cleared. Given that infections with P. falciparum are often polyclonal (23), patients with higher parasitemias could also have a greater chance of carrying a resistant clone. However, we showed that the association between mutations and PCT remained significant after adjustment for parasitemia level at admission. We also adjusted by sulfadoxine and pyrimethamine concentrations 24 hours after therapy and found that mutations remained strongly associated with PCT.
The effect of poor nutritional condition on treatment response was also explored, and we found that only extremely low concentrations of plasma iron (diagnostic of iron deficiency anemia) were associated with a longer PCT. However, a multivariate analysis showed that this effect might be partially or totally explained by initial high parasitemia. Other nutritional indicators evaluated were not found to be associated with longer PCT. It is possible that evaluation of these indicators previous to the development of the malaria episode would give more precise estimates of nutritional condition, given that acute disease may alter some of these parameters. If this is true, misclassification of nutritional status might partly explain the lack of association.
Ideally, malaria treatment should result in gametocyte clearance to reduce transmission. This is particularly important in P. falciparum because the gametocytes of this malaria species have a long lifespan of up to 24 days (24). Treatment with SP has been reported to increase the proportion of gametocytes detected in blood smears of P. falciparum-infected patients (25). We found that infections with DHFR mutations have higher transmission potential as measured by gametocytemia. Transmission potential also increased with each additional day that subjects took to clear parasitemia after treatment with SP. Environmental factors (i.e., the addition of fresh erythrocytes) have been shown to influence the rate at which P. falciparum erythrocytic parasites differentiate to sexual stages (26), and "stress" is also thought to cause P. falciparum to commit to sexual development (27). Therefore, our findings indicating that gametocytemia is more frequent and of higher density in persons with longer PCT may be partially explained by the fact that PCT is a measure of the time that viable parasites are exposed to drug treatment and other stress factors.
We found that DHFR mutations were associated with longer PCT (in the range of 13 days) and the presence of gametocytes (from day 7 on). The most likely mechanism is that DHFR mutations, even if insufficient to permit the parasite to survive drug treatment, prolong parasite survival under drug pressure. This is reflected by the PCT and manifested by increased gametocytemia. Even though in our analysis DHFR mutations remained significantly associated with gametocytemia after adjustment for duration of PCT, this is probably due to the relative insensitivity of PCT as a measure of parasite survival, particularly when PCT is measured at 24 hourly intervals. In addition, failure to adjust for a factor that causes both longer PCT and increased gametocyte carriage would result in a residual association of the mutations and gametocytemia in the model we adjusted by PCT (28). While we adjusted for a number of likely factors, we did not directly measure or adjust for host immunity, which is a potential common cause of both longer PCT and gametocyte carriage. Age is frequently used as a surrogate for immunity in high-transmission settings such as Africa. We did not see an association between age and PCT or gametocytemia in this study, but in relatively low-transmission settings such as the Pacific coast of Colombia, age is likely to be a poor predictor of immunity, as evidenced by the wide age range of our symptomatic subjects.
Our data suggest that even in a setting where an antimalarial drug is highly efficacious and the parasite mutations that confer high-level resistance are absent, low-level drug resistance mutations may contribute to the potential for the transmission of P. falciparum and the spread of resistance. The prolonged parasite clearance associated with low-level DHFR mutations raises the possibility that increasing PCT could serve as a useful early warning sign of developing resistance before treatment failures become common. Our findings highlight the need to anticipate drug resistance before it arrives and to develop strategies for deterring the evolution and spread of resistance. Antimalarial drugs and drug combinations designed to eliminate both asexual and sexual parasites may deserve priority not only because they will reduce malaria transmission but also because they will reduce the spread of drug resistance in its earliest stages.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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