INTRODUCTION
Resistance of Plasmodium falciparum to antimalarial drugs is one of the most serious threats to the control of malaria.1, 2 Due to widespread chloroquine resistance many countries such as Malawi, Tanzania, and Kenya have changed the first-line antimalarial drug to sulfadoxine/pyrimethamine (S/P). Unfortunately, the clinical efficacy of S/P is diminishing as a consequence of the development of P. falciparum resistance to S/P, possibly rendering a limited lifespan of S/P.
Monitoring the prevalence of molecular markers of S/P resistance may be a useful adjunct to in vivo resistance mapping. In vitro, point mutations in codons 51, 59, 108, and 164 in the P. falciparum dihydrofolate reductase gene (dhfr) provide resistance to pyrimethamine.3– 5 Likewise, mutations in codons 436, 437, 540, 581, and 613 of the dihydropteroate synthetase gene (dhps) mediate resistance to sulfadoxine.6, 7 Certain combinations of mutations in dhfr and dhps are necessary to ensure survival of P. falciparum after S/P treatment in vivo. Triple mutations in dhfr codons 51, 59, and 108 alone or together with a single or a double mutation in dhps is probably necessary.8– 12 However, a universal marker of S/P resistance has not been identified, possibly because variations in host immunity and transmission intensity are important confounding factors.8 Introduction of insecticide-treated nets (ITNs) reduces malaria morbidity by lowering the transmission intensity,13– 17 and may have an impact on resistance as well.18, 19
In two neighboring villages, Magoda and Mpapayu, in the northeastern part of Tanzania, malaria transmission is constantly very intense. In 1993, the inhabitants of Magoda village participated in a study that investigated the prophylactic effect of weekly pyrimethamine/dapsone (Maloprim®; Glaxo-SmithKline, West Uxbridge, United Kingdom) on malaria morbidity, and provided S/P to the village in cases of treatment failures.20 The use of Maloprim for malaria prophylaxis and SP for treatment of malaria in children was associated with high levels of cross-resistance to S/P observed during the Maloprim trial20 and in subsequent years.21, 22 Furthermore, during the current bed net trial, S/P was used as a first-line antimalarial in children less than five years old in both communities.
The prevalence of mutations in the dhfr and dhps genes was high in both villages as well22 (Alifrangis M, unpublished data). The main aim of this study was to assess the short-term impact of ITNs on dhfr and dhps gene mutations. The ITNs were distributed in Magoda village in 1998, while Mpapayu did not receive ITNs until two years later.
MATERIALS AND METHODS
Study population.
The study was carried out in Magoda and Mpapayu villages in the Muheza district of Tanzania in November–December 1998, 1999, and 2000. The distance between the villages is approximately 2 km. Permethrin-treated nets were distributed to all the households in Magoda village in December 1998, while Mpapayu village received delta-methrin-treated nets in March 2001. The ITNs were re-impregnated twice a year. Parents/guardians of all children between 0.5 and 5 years of age were asked for consent to participate in the study; approximately 90% of the total population of children did each year. Blood samples were collected by finger prick and examined for malaria parasites using Giemsa-stained thin and thick blood films. Additionally, 300 μL of blood were collected into EDTA-containing Eppendorf tubes (Radiometer, Roedovre, Denmark) and centrifuged at 2,000 rpm for 10 minutes. The packed red blood cells (RBCs) were stored at −20°C before being transported on dry ice to the Institute for Medical Microbiology and Immunology in Copenhagen, Denmark for genotype examination. Packed RBCs from 172 and 103 children in 1998, 173 and 100 children in 1999, and 170 and 100 children in 2000 from Magoda and Mpapayu villages, respectively, were collected for dhfr/dhps genotyping. For a few samples, the corresponding recordings of temperature or packed cell volume (PCV) were not available and these donors were excluded in the analysis of the in vivo data (less than 10%).
Extraction of DNA and polymerase chain reaction (PCR) genotype analysis.
Fifty microliters of packed RBCs were incubated overnight in 250 μL of proteinase K solution at 37°C (1 mM EDTA, 15 mM Tris, 150 mM NaCl, 1% sodium dodecyl sulfate, 100 μg/mL of proteinase K) (catalog no. 744 723; Roche, Hvidovre, Denmark). The samples were then extracted with phenol/chloroform and precipitated with ethanol as described by Sambrook and others.23 One microliter of the extracted DNA suspension was added to the PCR mixture. The degree of clonality of infections was estimated by PCR typing of the polymorphic regions of the P. falciparum merozoite surface protein 1 (msp1) and msp2 genes as described by Snounou and others.24 Only a subset of samples randomly selected was tested. For the dhfr and dhps genotyping, a nested PCR-restriction fragment length polymorphism described by Duraisingh and others25 was applied. However, the two outer dhfr and dhps PCRs were multiplexed into a single reaction. In addition, a nested dhps PCR targeting codon 540 specifically was designed (codon 540-K/, PCR product of 126 basepairs, same reaction conditions as the other nested dhfr and dhps PCRs25). The codon 540 primer sequence was: 5′-GCATAAAAGAGGAAATCCA-CATACAATGGtT-3′. The lower case base is one mismatch engineered into the primer to provide a cleavage site for the restriction enzyme Mse I in detecting the codon 540 wild type (Lys).
Statistical analysis.
The chi-square test with Yates’ correction or Fisher’s exact test was applied to compare the genotypic prevalences. One-way analysis of variance using the Student-Newman-Keuls method or Kruskal-Wallis one-way analysis on ranks was used to detect differences between the years. P values less than 0.05 were considered significant. All calculations were performed using Sigmastat version 2.03 software (Jandel Scientific, San Rafael, CA).
RESULTS
Prevalence and density of P. falciparum infections and PCV.
The prevalence of microscopically detectable P. falciparum infections decreased significantly in both villages between 1998 and 1999 (P ≤ 0.001) (Figure 1A). No differences in prevalence were found when comparing the villages. In Magoda, the mean density of P. falciparum infections was significantly lower in 1999 than in 1998 (P < 0.001) and remained low in 2000 (Figure 1B). Furthermore, the mean parasite density was lower in Magoda than in Mpapayu in 1999 and 2000 (P = 0.03 and P = 0.21 for Magoda versus Mpapayu in 1999 and 2000, respectively). The mean PCV (Figure 1C), a marker of anemia, increased between 1998 and 1999 in both villages, and it continued to increase in Magoda between 1999 and 2000 (P < 0.05). Furthermore, there was a significantly higher mean PCV level in Magoda than in Mpapayu in 2000 (P < 0.05). There was no difference in age, sex ratio, or mean temperature between the years and between the villages.
Mean clone multiplicity in the villages in 1998–2000.
The mean number of clones per infection estimated by PCR genotyping on polymorphic regions of msp1 and msp2 is shown in Table 1. The mean clone multiplicity decreased significantly in Magoda from 1998 to 2000 for msp1, msp2, and the multiplicity of infection (MOI, the minimal number of clones per infection when combining msp1 and msp2 data) (P < 0.05). In Mpapayu, no significant differences were observed between the years (P = 0.61, P = 0.29, and P = 0.45 for msp1, msp2, and MOI, respectively). A comparison between the villages for the three years showed a significantly lower clone multiplicity in Magoda versus Mpapayu in 2000 for msp1 (P = 0.002) and MOI (P = 0.011). For msp2, a significantly lower clone multiplicity was found in Magoda versus Mpapayu in 1999 and 2000 (P = 0.04 for both comparisons).
Prevalence of dhfr genotypes in the villages in 1998–2000.
The genotypic prevalence of codons 51, 59, and 108 in the dhfr gene in P. falciparum infections in the two villages in 1998, 1999 and 2000 are shown in Figure 2. The infections are divided into wild type and mutant type parasite populations. The remaining infections were expressing both wild and mutant type (mixed) genotypes (not shown in Figure 2, but included in the following statistical analysis where mutant type infections including mixed infections are compared with wild types). In Magoda, the year 2000, the prevalence of codon 51 and 108 wild types (Figure 2A and C) was significantly higher than in the two preceding years (P ≤ 0.001 for both codons 51 and 108). For codon 59 (Figure 2B), the significant increase in codon 59 wild types was seen between 1998 and 1999 rather than between 1999 and 2000 (P = 0.003 for 1998 versus 1999 and P = 0.119 for 1999 versus 2000). Surprisingly, no evident differences in the prevalences of the three codons were observed for Mpapayu between the three years. Conversely, the prevalence of these wild types was significantly higher in Magoda when compared with Mpapayu in 2000 (P = 0.002, P ≤ 0.001, and P = 0.002 for comparison of Magoda and Mpapayu in 2000 for codons 51, 59, and 108, respectively).
The prevalence of combinations of dhfr genotypes is shown in Figure 2D. The infections are divided into parasite populations expressing true triple wild types at codons 51, 59, and 108 or true triple mutant types at all three codons. The remaining infections are mixed genotypes at one or more codons (not shown in Figure 2, but included in the following statistical analysis where true triple mutant type infections including mixed infections are compared with true triple wild types). The prevalence of true triple wild types increased significantly in Magoda in 2000 (P ≤ 0.001). In Mpapayu, there was no evident difference in the prevalence of true triple wild types. Conversely, the prevalence of true triple mutant type infections increased significantly in 2000 (P ≤ 0.001, for true triple wild type infections including mixed infections versus true triple mutant types). Accordingly, a significant higher proportion of true triple wild types was found in Magoda when compared with Mpapayu in 2000 (P ≤ 0.001).
Prevalence of dhps genotypes in the villages in 1998–2000.
The genotypic prevalence of codons 436, 437, and 540 in the dhps gene between 1998 and 2000 are shown in Figure 3. For codon 436 (Figure 3A) only Ser-436 or Ala-436 was found and no clear differences were observed from year to year in the two villages or between the two villages within the same year. For codon 437 (Figure 3B), a significant increase in the prevalence of mutant type infections in Magoda in 2000 was observed (P ≤ 0.001). For codon 540 (Figure 3C), there was a significant increase in the prevalence of wild type infections in Magoda and Mpapayu from year to year (P ≤ 0.001 and P = 0.049 for Magoda and Mpapayu, respectively).
DISCUSSION
In Tanzania, increasing levels of chloroquine resistance necessitated changing the first-line antimalarial drug to S/P by year 2001. Unfortunately, in vivo resistance to S/P is already evident in Tanzania26, 27 and may render this drug combination useless within a few years. Lowering the level of transmission may be a method to indirectly increase S/P susceptibility by lowering drug use and, thus, drug pressure.18, 28
In December 1998, ITNs were introduced in Magoda village while the nearby village of Mpapayu first received ITNs in the beginning of 2001. From our data shown in Figure 1, it appears that an effect of ITN on parasite prevalence and density as well as PCV in Magoda was evident in 2000, when a lower prevalence of P. falciparum infections and a lower mean P. falciparum density were found in Magoda versus Mpapayu. Furthermore, the overall PCV level increased between 1998 and 1999 in both villages, but continued to increase only in Magoda in 2000.
The annual rainfall measured in the neighboring village of Masaika varied over the three years and the area received markedly less rain in 1999 (Rwegohora T, unpublished data). This will affect the neighboring villages, similarly lowering the transmission and possibly the main reason for the lower prevalence of P. falciparum infections seen in both villages in 1999, but is hardly the cause of alterations only seen in Magoda in 2000. Furthermore, the clone multiplicity of P. falciparum infections, measured by the mean number of P. falciparum clones using the polymorphic genes msp1 and msp2 only decreased significantly in Magoda, possibly as a result of decreased transmission intensity due to ITNs.
Apparently, ITNs seemed to have an effect on the composition of P. falciparum dhfr genotype infections in children less than five years of age in Magoda compared with those in Mpapayu. In all the polymorphic codons of dhfr (51, 59, and 108), the prevalence of wild type infections in Magoda increased from 1998 to 2000 and when combined, the prevalence of true dhfr wild type infections (wild type at codons 51, 59, and 108) increased significantly from 1999 to 2000. Furthermore, the prevalence of true triple wild type infections was significantly higher in Magoda compared with Mpapayu in 2000. However, for dhps genotypes, only marginal differences were observed. Interestingly, for the past seven years we have annually measured the prevalence of dhfr and dhps mutations as part of our annual S/P efficacy trials, and the occurrence of true triple wild types in the dhfr gene has constantly been less than 2%22 (Alifrangis M, unpublished data). The overall alteration seen in the dhfr genotypes are possibly a result of a decrease in P. falciparum clone multiplicity since the overall occurrence of mixed wild/mutant type infections generally decreased significantly from 1998 to 2000, especially in Magoda. This is further confirmed by the clone multiplicity data.
The accumulation of mutations in the dhfr and dhps genes in response to increasing S/P drug pressure has been reported to be stepwise and occurs first in the dhfr gene, where codon 108 is initially mutated, followed by mutations in codon 51 and/or codon 59.5 Changes in the dhps gene follow, where the mutation in codon 437 precedes the other mutations in this gene.10 When susceptibility to S/P in vivo is restored, this may proceed in the same manner: wild types in dhfr first appear, followed by changes in dhps, possibly explaining the sudden occurrence of triple dhfr wild type infections, while only minor changes in dhps polymorphisms are seen. Alternatively, since the role of mutations in dhps in conferring resistance to S/P is still debated, other mechanisms of resistance may explain the lack of changes in the dhps gene.
The survival of a drug-resistant mutant in a parasite population is strongly dependent on high drug pressure and a high transmission rate.29 The intervention with ITNs presumably lowers transmission and thereby reduces the prevalence of infections and thus the number of clinical attacks. Consequently, the use of S/P is reduced, decreasing the S/P drug pressure.18 In turn, this presumably selects for wild type parasites, and eventually mediates a change in dhfr genotype composition as observed, followed by an increased susceptibility to S/P. Accordingly, Mackinnon and Hastings simulated by mathematical modeling that lowering transmission rate reduced the spread of drug resistance in areas with high clone multiplicity and drug pressure and suggested bed nets as means of control.19
It could also be speculated that since infections in older children generally are asymptomatic, the survival advantage of mutated dhfr/dhps genotype populations is presumably limited, possibly explaining why age has been found to be inversely related to the prevalence of mutant genotypes.30 In infants and younger children, however, mutated parasites have an evident survival advantage and may consequently be more prevalent in this age group.
Interventions with ITNs protect mainly infants and young children from becoming infected31 probably because they are kept under nets from early evening compared with older children. When ITN intervention reduces transmission, mainly the parasite populations otherwise causing malaria in children less than five years old will be affected. The ITNs may thereby remove a greater proportion of these mutant parasite populations from circulating and thereby cause an overall reduction in prevalence of mutant genotypes. If our observations can be reproduced in other holoendemic areas with high levels of S/P resistance, it may have a major public health impact: ITNs may halt and even reverse the development of P. falciparum resistance to S/P.
Clone multiplicity analysis of Plasmodium falciparum in Magoda and Mpapayu, Tanzania in 1998–2000*
| Magoda | Mpapayu | |||||
|---|---|---|---|---|---|---|
| 1998 | 1999 | 2000 | 1998 | 1999 | 2000 | |
| * Average number of clones per infection estimated by polymerase chain reaction (PCR) genotyping on polymorphic regions of merozoite surface protein 1 (msp1) and msp2 with 95% confidence intervals in parentheses. MOI = multiplicity of infection, the minimal number of clones per infection when combining msp1 and msp2 data. A subset of samples were randomly selected for PCR genotyping: n = 80, 64, and 60 for Magoda village in 1998, 1999, and 2000, respectively, and n = 48, 44, and 40 for Mpapayu village in 1998, 1999, and 2000, respectively. Of these, a subset was PCR positive. | ||||||
| PCR positive | 74 | 51 | 48 | 47 | 37 | 37 |
| Msp1 | 3.4 (3.1–3.8) | 2.5 (2.1–3.0) | 1.8 (1.5–2.1) | 3.2 (2.8–3.7) | 2.9 (2.5–3.5) | 2.8 (2.2–3.4) |
| Msp2 | 3.0 (2.7–3.4) | 2.5 (2.1–3.0) | 2.1 (1.8–2.5) | 3.5 (3.1–4.1) | 3.3 (2.7–3.9) | 2.8 (2.3–3.4) |
| MOI | 3.9 (3.6–4.2) | 2.9 (2.4–3.5) | 2.3 (1.9–2.8) | 3.9 (3.4–4.5) | 3.5 (2.9–4.1) | 3.2 (2.6–4.0) |
Prevalence and density of Plasmodium falciparum infections and the geometric mean (GM) packed cell volume (PCV) in children less than five years old in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. The prevalence (A) and mean density of P. falciparum infections per microliter (B) are based on blood slide examination by microscopy (C). Error bars show 95% confidence intervals. N = 161, 165, and 170 for Magoda village in 1998, 1999 and 2000, respectively, and 98, 93, and 100 for Mpapayu village in 1998, 1999 and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence and density of Plasmodium falciparum infections and the geometric mean (GM) packed cell volume (PCV) in children less than five years old in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. The prevalence (A) and mean density of P. falciparum infections per microliter (B) are based on blood slide examination by microscopy (C). Error bars show 95% confidence intervals. N = 161, 165, and 170 for Magoda village in 1998, 1999 and 2000, respectively, and 98, 93, and 100 for Mpapayu village in 1998, 1999 and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence and density of Plasmodium falciparum infections and the geometric mean (GM) packed cell volume (PCV) in children less than five years old in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. The prevalence (A) and mean density of P. falciparum infections per microliter (B) are based on blood slide examination by microscopy (C). Error bars show 95% confidence intervals. N = 161, 165, and 170 for Magoda village in 1998, 1999 and 2000, respectively, and 98, 93, and 100 for Mpapayu village in 1998, 1999 and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. A, Codon 51. B, Codon 59. C, Codon 108. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. D, Codons 51, 59, and 108 combined into true triple mutant types (black circles) and true triple wild types (white squares). The remaining children were infected with parasite populations with mixed genotypes (including infections with one or two wild types in codons 51, 59, and 108). Error bars show 95% confidence intervals. The number of samples that were PCR positive was 149, 116, and 111 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 84, and 75 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. A, Codon 51. B, Codon 59. C, Codon 108. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. D, Codons 51, 59, and 108 combined into true triple mutant types (black circles) and true triple wild types (white squares). The remaining children were infected with parasite populations with mixed genotypes (including infections with one or two wild types in codons 51, 59, and 108). Error bars show 95% confidence intervals. The number of samples that were PCR positive was 149, 116, and 111 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 84, and 75 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu villages in Tanzania in 1998, 1999, and 2000. A, Codon 51. B, Codon 59. C, Codon 108. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. D, Codons 51, 59, and 108 combined into true triple mutant types (black circles) and true triple wild types (white squares). The remaining children were infected with parasite populations with mixed genotypes (including infections with one or two wild types in codons 51, 59, and 108). Error bars show 95% confidence intervals. The number of samples that were PCR positive was 149, 116, and 111 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 84, and 75 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu in 1998, 1999, and 2000. A, Codon 436. B, Codon 437. C, Codon 540. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. Error bars show 95% confidence intervals. The number of samples that were PCR positive was 148, 110, and 113 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 78, and 73 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu in 1998, 1999, and 2000. A, Codon 436. B, Codon 437. C, Codon 540. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. Error bars show 95% confidence intervals. The number of samples that were PCR positive was 148, 110, and 113 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 78, and 73 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Prevalence of Plasmodium falciparum dihydrofolate reductase genotypes in Magoda and Mpapayu in 1998, 1999, and 2000. A, Codon 436. B, Codon 437. C, Codon 540. Black circles = mutant type; white squares = wild type. Infections with mixed genotypes (both wild and mutant genotypes) are not shown. Error bars show 95% confidence intervals. The number of samples that were PCR positive was 148, 110, and 113 for Magoda village in 1998, 1999, and 2000, respectively, and 90, 78, and 73 for Mpapayu village in 1998, 1999, and 2000, respectively.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 3; 10.4269/ajtmh.2003.69.238
Authors’ addresses: Michael Alifrangis and Insaf F. Khalil, Panum Institute, Institute of Medical Microbiology and Immunology, Building 24.2, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Martha M. Lemnge, Method D. Segeja, and Stephen M. Magesa, National Institute for Medical Research, Amani Research Centre, PO Box 4, Amani, Tanga, Tanzania. Anita M. Rønn and Ib C. Bygbjerg, Institute of Public Health, University of Copenhagen, Nørre Alle 4-6, 2200 Copenhagen N, Denmark.
Acknowledgments: We are grateful to the people of Magoda and Mpapayu villages, the village helpers and the technicians, Julius Mhina and Jumaa Akida (National Institute for Medical Research, Amani, Tanzania) for assisting with the fieldwork in Tanzania. We thank laboratory technicians Jimmy Weng and Gitte Hoff Jensen for excellent technical assistance in performing the polymerase chain reaction–restriction fragment length polymorphism. We also thank Theophil Rwegohora (National Institute for Medical Research, Bombo Field Station, Tanga, Tanzania) for providing annual rainfall data for the neighboring village of Masaika, Tanzania, and Thor G. Theander, Lars Hviid (Centre for Medical Parasitology, Copenhagen, Denmark), and Anders Björkman (Karolinska Institute, Stockholm, Sweden) for helpful comments on this manuscript.
Financial support: This study was supported by a grant from the Research Council of the Danish International Development Agency (DANIDA RUF, grant no. 90892). The study in Tanzania was supported by DANIDA grant no. 104.Dan.8L as part of a Tanzanian-Danish collaboration under the Enhancement of Research Capacity (ENRECA) program.
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