Abstract

BackgroundEnormous amounts of drugs were used to contain the outbreak of infectious diseases in areas of India affected by the tsunami in December 2004. The impact of this drug use on the Plasmodium falciparum population needs to be investigated

MethodsThe nucleotide sequence of the pfcrt, pfdhps and pfdhfr genes was determined for 229 clinical P. falciparum isolates collected from patients on Car Nicobar Island at 6 different time points between May 2004 and May 2008

ResultsOver time, there was an increase in the proportion of the P. falciparum population that had mutations in the pfcrt, pfdhps and pfdhfr genes associated with higher levels of chloroquine, sulfadoxine, and pyrimethamine resistance, respectively. However, the parasites collected during October 2005 had mutations associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance (a rare combination), as well as a novel K540N mutation in P. falciparum dihydropteroate synthetase (PfDHPS). The emergence of this parasite population coincided with the widespread use of an additional antifolate drug, trimethoprim-sulfamethoxazole, to treat other infections during January–March 2005. Molecular modeling revealed that the sulfadoxine binding affinity of the new PfDHPS triple mutant A436 G437 N540A581A613 was similar to that of A436 G437 E540A581A613 (bold type indicates mutated amino acids)

ConclusionsThe use of 2 antifolate drugs in combination should be avoided to prevent the selection of parasites with newer mutations and altered drug susceptibilities

Chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) are the antimalarial drugs most commonly used to treat Plasmodium falciparum malaria. Their modes of action are different. CQ accumulates in the food vacuole of the malaria parasite, thus changing its pH and interfering in the formation of hemozoin [1]. However, the parasite has developed CQ efflux mechanisms, and resistance to this drug has emerged [2]. Some of the proteins located in the food vacuole membrane, including P. falciparum chloroquine resistance transporter (PfCRT), assist the parasite in the efflux process [3]. Certain point mutations in PfCRT have been shown to be associated with CQ resistance [4]. On the other hand, SP interferes with the parasite’s folate biosynthesis pathway by inhibition of the enzymes dihydropteroate synthetase (DHPS, inhibited by sulfadoxine) and dihydrofolate reductase (DHFR, inhibited by pyrimethamine). However, the parasite has developed resistance to these antifolate drugs as a result of certain point mutations at key amino acid positions in these enzymes [5] CQ and SP resistance can thus be monitored in the field by evaluating point mutations in the pfcrt, pfdhfr and pfdhps genes of clinical P. falciparum isolates [6, 7]

The December 2004 tsunami disaster in the Indian Ocean devastated several Southeast Asian countries and several costal areas of India. Large tidal waves not only killed large numbers of people and destroyed their homes, but also caused waterlogging and other conditions conducive to the outbreak of several infectious diseases. To prevent these outbreaks, several preventive measures, including mass drug-treatment programs, were implemented by the Indian government in its affected territories. Although these strategies were, to a certain extent, successful at containing disease outbreaks, their effect on the genomes of the existing microorganisms remains unexplored. To assess the selection pressure exerted by CQ and SP on the affected P. falciparum parasite population of Car Nicobar Island, we investigated mutations in the parasite’s relevant marker genes over a 4-year period. Surprisingly, we found that the isolates collected within 1 year after the tsunami exhibited an allelic frequency of PfDHFR and PfDHPS mutations that differed from that of isolates collected before the tsunami, as well as a novel K540N mutation in PfDHPS. However, the isolates collected >1 year after the tsunami showed normal allelic frequency for the PfDHFR and PfDHPS mutations associated with a higher level of drug resistance

Materials and Methods

Study site and sample collectionCar Nicobar Island is one of the Andaman and Nicobar group of islands in the Indian Ocean. It is situated in the southeast corner of the island group at 6–10° north latitude and 92–94° east latitude in the Bay of Bengal (figure 1). Because of its flat surface and low elevation relative to the other islands in the group, it was the island worst affected during the tsunami disaster in December 2004. The majority of the population are Nicobarese tribe members of Mongol descent

Figure 1

Map of India and adjoining countries showing the location of Car Nicobar Island in the Andaman and Nicobar group of islands

Figure 1

Map of India and adjoining countries showing the location of Car Nicobar Island in the Andaman and Nicobar group of islands

In accordance with India’s national drug policy, all patients with a fever (temperature >37°C in patients attending a malaria clinic or in the field) were treated with the presumptive dose of antimalarial drugs (adult dose, 600 mg CQ and 45 mg primaquine); radical treatment (adult dose, 600 mg and 300 mg CQ on the second and third day, respectively, after the first dose) was given to patients who were found to be positive for the malaria parasite by light microscopy. Patients with malaria who did not respond to the radical treatment and continued to show parasitemia as well as fever were then treated with SP (adult dose, 1500 mg sulfadoxine and 75 mg pyrimethamine as a single dose). Patients who did not respond to SP after another 48–72 h were treated with quinine (adult dose, 600 mg quinine sulfate 3 times per day for 7 consecutive days) or artesunate (60 mg 2 times per day for the first day and 60 mg 1 time per day for the next 4 days, intravenously administered)

During January–March 2005, a large number of patients received both SP and trimethoprim-sulfamethoxazole (TS) because the latter was widely used to treat bacterial infections during this time (adult dose, 160 mg trimethoprim and 800 mg sulfamethoxazole 2 times per day for 5 consecutive days). We obtained informed oral consent from patients positive for the malaria parasite and collected heparinized blood samples of 100–200 μL. Institutional ethical guidelines for blood collection were followed. We collected blood samples from patients infected with P. falciparum at the following 6 time points: May–October 2004 (group A), January–February 2005 (group B), October 2005 (group C), February 2006 (group D), May–July 2007 (group E), and April–May 2008 (group F). The samples were stored at −80°C until they were analyzed

Polymerase chain reaction (PCR)Genomic DNA was extracted from P. falciparum–infected blood by use of the AccuPrep Genomic DNA Extraction Kit (Bioneer Corporation), in accordance with the manufacturer’s instructions. This genomic DNA was then used to amplify the pfdhfr gene and regions of the pfdhps and pfcrt genes. Primer sequences and PCR amplification conditions have been described elsewhere [8–10]. The PCR product of the pfdhfr gene covered the entire coding region, whereas the PCR products of the pfdhps and pfcrt genes covered codons 392–637 and 44–177, respectively

Nucleotide sequencingThe PCR products were purified from agarose gel by use of the QIAEX-II Gel Extraction Kit (QIAGEN), in accordance with the manufacturer’s instructions. We used 50–250 ng of the gel-purified product for PCR sequencing with the ABI Big Dye Terminator Ready Reaction Kit (version 3.1; PE Applied Biosystems), as described elsewhere [8]. Templates were purified and sequenced on the ABI Prism 310 Genetic Analyzer (PE Applied Biosystems)

Sequence analysisThe sequences we obtained were translated by using the translation tool available online at the Expert Protein Analysis System proteomic server (http://www.expasy.org), and these translated sequences were then aligned by use of the online multiple sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/clustalw)

Computer modeling and dockingHomology modeling was carried out using 3-dimensional structures of DHPS with 33% identity from Bacillus anthrax (PDB ID-1TX0) [11] and Mycobacterium tuberculosis (PDB ID-1EYE) [12] templates with the inclusion of pterin monophosphate (PtP) and the Mg2+ ion. Mutants were generated by altering the corresponding residues; they were minimized separately in the presence of PtP, Mg2+, and sulfadoxine and validated with PROCHECK [13]. The docking of the minimized sulfadoxine was guided by the orientation of the substrate, p-amino benzoic acid. An empirical scoring function, the LUDI energy estimate [14], was used to predict binding affinity. Molecular dynamics simulation was carried out in 3 stages in the fully hydrated model for each mutant to check the stability and binding of the docked ligand. The software package Discovery Studio (version 1.7; Accelrys) and its modules were used

Statistical analysisThe χ2 test was used to detect significant differences between the 2 groups of P. falciparum isolates (i.e., those collected before the tsunami and those collected after it), with statistical significance defined as P<.05

Results

A total of 315 blood samples were obtained from P. falciparum–positive patients on Car Nicobar Island at 6 different time points over a period of 4 years (May 2004 to May 2008). Samples were subjected to PCR amplification and sequencing of the pfcrt, pfdhfr and pfdhps genes, either in part or in full. We were able to analyze sequence data from 229 clinical isolates

PfCRTA total of 184 isolates provided a clear sequence of exon 2 of the pfcrt gene, which covers the important codons 72–76. The majority of the isolates (117 [63.58%] of 184 isolates) had the Southeast Asian genotype (C72V73 I74 E75 T76; bold type in genotype sequences indicates mutation), and only 47 (25.54%) of the isolates had the South American allele ( S72V73M74N75 T76). The allelic frequency of these 2 genotypes was similar among the group A isolates collected during 2004 (P=.524) (figure 2). However, the allelic frequency changed in isolates collected during subsequent years, and C72V73 I74 E75 T76 became the predominant genotype (97 [70.80%] of 137). Compared to group A isolates, the number of isolates with multiple mutations was significantly larger in the other isolate groups (P<.002)

Figure 2

Distribution of Plasmodium falciparum genotypes for chloroquine resistance transporter (PfCRT) among isolates collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar Island in India. Bold type in key indicates mutated amino acids

Figure 2

Distribution of Plasmodium falciparum genotypes for chloroquine resistance transporter (PfCRT) among isolates collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar Island in India. Bold type in key indicates mutated amino acids

PfDHFRWe sequenced the pfdhfr gene from 200 P. falciparum isolates. None of the isolates showed A16V and S108T mutations, which are related to the cycloguanil resistance, in PfDHFR. This is because cycloguanil is not used as an antimalarial drug in India. The majority of isolates (137 [68.50%] of 200 isolates) contained quadruple mutations and had the A16 I51 R59 N108 L164 genotype. Among the 48 P. falciparum isolates collected during 2004, the allelic frequency of the A16 I51 R59 N108 L164 genotype was higher (41 isolates [85.42%]) than that of the A16N51 R59 N108I164 genotype (2 [4.17%] of 48) (P<.001). However, compared with group A isolates, the group C isolates (collected during October 2005; n=55) showed a significant change with respect to the allelic frequency of the A16N51 R59 N108I164 genotype (P<.001) and the A16 I51 R59 N108 L164 genotype (P<.001) (figure 3). Nevertheless, the allelic frequency of the A16 I51 R59 N108 L164 genotype increased in isolates obtained after October 2005

Figure 3

Distribution of Plasmodium falciparum genotypes for dihydrofolate reductase (PfDHFR) among isolates collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar Island in India. Bold type in key indicates mutated amino acids

Figure 3

Distribution of Plasmodium falciparum genotypes for dihydrofolate reductase (PfDHFR) among isolates collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar Island in India. Bold type in key indicates mutated amino acids

PfDHPSA total of 208 P. falciparum isolates provided a clear pfdhps gene sequence that covered codons 436, 437, 540, 581, and 613. Eleven different PfDHPS genotypes were observed in these isolates; the A436 G437 E540A581A613 genotype was more common (69 [33.17%] of 208 isolates) than the others. A significantly greater number of group A isolates had single mutations (P<.001) and double mutations (P<.001), compared with isolates from the other groups collected later. Conversely, the number of group A isolates with triple mutations was significantly smaller than the number of such isolates in the other groups (P<.001). The allelic frequency of genotypes was also different when group A and the rest of the isolates were compared; genotype A436 G437K540A581A613 was more common among group A isolates (21 [42.00%] of 50), whereas genotype A436 G437 E540A581A613 was more common among the rest of the isolates (63 [39.87%] of 158) (table 1). Certain genotypes were observed only in group C ( A436 G437K540 G581A613 and A436 G437 N540A581A613) and others only in group D (S436A437 N540A581A613 and A436 G437 E540 G581A613), whereas all of the other genotypes were observed in >1 group

Table 1

Distribution of the genotypes for dihydropteroate synthetase among the Car Nicobar Plasmodium falciparum isolates, according to isolate group

Table 1

Distribution of the genotypes for dihydropteroate synthetase among the Car Nicobar Plasmodium falciparum isolates, according to isolate group

Combined PfDHFR-PfDHPS mutationsSequencing information for all the desired codons of both genes was successfully obtained from 175 P. falciparum isolates. There were 28 different combined PfDHFR-PfDHPS 2-locus genotypes (table 2). Genotype A16 I51 R59 N108 L164 A436 G437K540A581A613 was predominant among the group A isolates (14 [34.15%] of 41 isolates). Conversely, genotype A16 I51 R59 N108 L164 A436 G437 E540A581A613 was predominant among all other groups of isolates except group C. Indeed, the most common genotype in group C, A16N51 R59 N108I164 A436 G437 N540A581A613, was not seen in any other group of isolates

Table 2

Distribution of the 2-locus Plasmodium falciparum genotypes for dihydrofolate reductase and dihydropteroate synthetase (PfDHFR-PfDHPS) among the Car Nicobar isolates, according to isolate group

Table 2

Distribution of the 2-locus Plasmodium falciparum genotypes for dihydrofolate reductase and dihydropteroate synthetase (PfDHFR-PfDHPS) among the Car Nicobar isolates, according to isolate group

The total number of PfDHFR-PfDHPS 2-locus mutations among these 175 isolates varied from 2 to 8 (figure 4). While the majority of group A isolates (20 [48.78%] of 41) contained six 2-locus mutations, the majority of isolates from the remaining groups (except group C) were found to contain seven 2-locus mutations. The majority of group C isolates (21 [44.68%] of 47), on the other hand, were found to contain only five 2-locus mutations

Figure 4

Total no. of 2-locus mutations in Plasmodium falciparum genotypes for dihydrofolate reductase and dihydropteroate synthetase (PfDHFR-PfDHPS) in each group of isolates. Isolates were collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar island in India

Figure 4

Total no. of 2-locus mutations in Plasmodium falciparum genotypes for dihydrofolate reductase and dihydropteroate synthetase (PfDHFR-PfDHPS) in each group of isolates. Isolates were collected at 6 different time points between May 2004 and May 2008 from patients on Car Nicobar island in India

Expected drug resistanceWe have previously shown that parasites with the PfCRT genotype C72V73 I74 E75 T76 are more resistant to CQ than are parasites with the S72V73M74N75 T76 genotype [8]. The increased frequency with which the C72V73 I74 E75 T76 genotype was observed over a period of 4 years indicates that there was selection pressure that resulted in an increase in the level of CQ resistance in the parasite population. Similarly, the increased number of PfDHFR-PfDHPS 2-locus mutations also indicates an increase in the level of SP resistance in the parasite population [9]. Considered together, the majority of the isolates collected during 2007–2008 clearly showed a greater number of mutations in the pfcrt, pfdhfr and pfdhps genes associated with a higher level of CQ and SP resistance than did the majority of isolates in group A, collected 4 years earlier. However, group C isolates were an exception to this pattern; they showed an increased number of the mutations that are associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance (a unique combination), as well as a novel K540N mutation in PfDHPS

Effect of the new PfDHPS mutation (K540N) on sulfadoxine resistanceIt has been experimentally shown that the level of sulfadoxine resistance increases with alterations in the amino acid residues S436A, A437G, and K540E of PfDHPS. Structural analysis of the drug’s potency with respect to different forms of PfDHPS was performed with the help of molecular modeling. Wild-type PfDHPS forms a greater number interactions with sulfadoxine (figure 5A) than do the mutated forms (figures 5B–5D), indicating that the wild type has the highest binding affinity for the drug. In the form with a single mutation, K540N (S436A437 N540A581A613), the hydrogen bond with the drug cannot be formed because of the shortening of the side chain. Consequently, the binding affinity for the drug decreases (figure 5B). In the triple mutant A436 G437 N540A581A613, the alteration at residue 437 results in shortening of the hydrophobic side chain to form the more supple glycine, thus imparting greater flexibility to the backbone. A loss of hydrophilicity occurs as a result of the modification at position 436, and the water-linked hydrogen bond can no longer be formed. The subsequent change in the backbone conformation results in a lower affinity for the drug at the active site. The drug is therefore no longer an effective inhibitor (figure 5C). In the triple mutant A436 G437 E540A581A613, the residue 540 is altered to form the more acidic and bulky glutamic acid (figure 5D). The resulting environmental difference induces a conformation change at the loop and thus increases resistance to the drug. The 2 novel PfDHPS mutants S436A437 N540A581A613 (single mutant) and A436 G437 N540A581A613 (triple mutant) show a predicted binding affinity of 4.07 and 90.2 μmol/L, respectively

Figure 5

Models of wild-type Plasmodium falciparum dihydropteroate synthetase (PfDHPS) and its mutated forms with docked sulfadoxine (SFD) (ball-and-stick models) showing the interactions of the drug with the protein. The hydrogen-bonded and mutated residues are indicated by sticks, whereas the dotted line represents the residues involved in van der Waals contacts. A Wild-type PfDHPS: SFD makes 1 indirect and 5 direct hydrogen-bonded interactions through a water molecule with the wild-type PfDHPS structure.B S436A437 N540A581A613 genotype: the mutated Asn 540 Oδ1 now forms a hydrogen bond with the backbone Asn 534 N, which results in the loss of this hydrogen bond with the drug. C A436 G437 N540A581A613 genotype: the 2 hydrogen-bonded interactions with the drug, in addition to the long range of hydrophobic interactions, are Asn 396 Oδ1 and Arg 608 N. D A436 G437 E540A581A613 genotype: Alterations at residues 436 and 437 are similar to the novel mutant A436 G437 N540A581A613, and thus the backbone at this loop in both the triple mutants adopts a similar conformation. The mutation K540E induces a conformational change in the loop, bringing about a slight change at the binding-site pocket that causes the drug to bind in a slightly different orientation. This loop is further stabilized by salt-bridge formation between Glu 540 Oɛ1 and Oɛ2 atoms with Arg 610 NH1 and His 584 Nδ1, respectively. The side chain of Arg 610 reorients itself to enable the hydrogen bond formation. Figures were drawn with PyMOL Molecular Viewer (DeLano Scientific)

Figure 5

Models of wild-type Plasmodium falciparum dihydropteroate synthetase (PfDHPS) and its mutated forms with docked sulfadoxine (SFD) (ball-and-stick models) showing the interactions of the drug with the protein. The hydrogen-bonded and mutated residues are indicated by sticks, whereas the dotted line represents the residues involved in van der Waals contacts. A Wild-type PfDHPS: SFD makes 1 indirect and 5 direct hydrogen-bonded interactions through a water molecule with the wild-type PfDHPS structure.B S436A437 N540A581A613 genotype: the mutated Asn 540 Oδ1 now forms a hydrogen bond with the backbone Asn 534 N, which results in the loss of this hydrogen bond with the drug. C A436 G437 N540A581A613 genotype: the 2 hydrogen-bonded interactions with the drug, in addition to the long range of hydrophobic interactions, are Asn 396 Oδ1 and Arg 608 N. D A436 G437 E540A581A613 genotype: Alterations at residues 436 and 437 are similar to the novel mutant A436 G437 N540A581A613, and thus the backbone at this loop in both the triple mutants adopts a similar conformation. The mutation K540E induces a conformational change in the loop, bringing about a slight change at the binding-site pocket that causes the drug to bind in a slightly different orientation. This loop is further stabilized by salt-bridge formation between Glu 540 Oɛ1 and Oɛ2 atoms with Arg 610 NH1 and His 584 Nδ1, respectively. The side chain of Arg 610 reorients itself to enable the hydrogen bond formation. Figures were drawn with PyMOL Molecular Viewer (DeLano Scientific)

Discussion

We have analyzed here a population of P. falciparum parasites in samples obtained from patients on Car Nicobar Island over a period of 4 years (i.e., from May 2004 to May 2008). We show that, compared with isolates obtained in 2004, a greater number of the isolates obtained in 2007–2008 harbored >2 mutations in pfcrt, pfdhfr and pfdhps genes and thus higher levels of CQ resistance and SP resistance (figures 2 and 3, and table 1). This change over time indicates continuous selection pressure exerted by drugs and is in agreement with our earlier observations regarding the parasite population of mainland India. [8,9]

However, we noticed an unusual selection of parasites among the group C isolates, which were collected during October 2005. In this group, the proportion of the parasite population with quadruple PfDHFR mutations (A16 I51 R59 N108 L164), which are associated with the highest level of pyrimethamine resistance, had declined, whereas the proportion of the population with double mutations (A16N51 R59 N108I164), which are associated with a lower level of pyrimethamine resistance, had expanded significantly (figure 3). Conversely, the proportion of the population in this group with triple PfDHPS mutations expanded, and the proportion with double mutations declined significantly, indicating that this group experienced greater selection pressure from sulfadoxine (table 1). Furthermore, continuation of this greater sulfadoxine pressure may have also resulted in selection for the parasite population in group D that contained the quadruple PfDHPS mutation ( A436 G437 E540 G581A613). This allelic frequency for PfDHFR and PfDHPS mutations is unusual because under normal circumstances, PfDHFR mutations occur first, followed by PfDHPS mutations [5, 9]. Therefore, under continuous selection pressure from SP, one would expect selection of parasites with more mutations in PfDHFR than in PfDHPS, unlike the observed allelic frequency of mutant genotypes in this population, which was the reverse

Why were parasites with increased sulfadoxine resistance and decreased pyrimethamine resistance selected in the group of isolates obtained after the 2004 tsunami (group C), even though both components of SP were given in combination? While searching for possible explanations, we found that, in addition to SP, the antifolate drug TS was also widely used during this period. This antifolate drug was used to treat bacterial infections (adult dose, 160 mg trimethoprim and 800 mg sulfamethoxazole twice per day for 5 consecutive days). According to rough estimates, >70% of patients with malaria were treated with both SP and TS during January–March 2005

It is known that both SP and TS can independently inhibit parasite growth since both drugs target the same parasite enzymes involved in the folate biosynthesis pathway [15]. Parasites probably use the same molecular mechanisms to develop resistance to both of these antifolate drugs [5, 16, 17]. Therefore, the malarial parasite can develop cross-resistance to SP and TS. It might be postulated that because of cross-resistance to the sulfa component of SP and TS, selection favored the parasite population with a greater number of PfDHPS mutations in this group of isolates obtained after the 2004 tsunami. However, the same argument does not explain the decline in the number of parasites with a greater number of PfDHFR mutations in the same group. It might be suggested here that cross-resistance for SP and TS exists, but it is not complete [18]. However, complete cross-resistance for the sulfa drugs as a result of PfDHPS mutations has been clearly established [17], whereas pyrimethamine and trimethoprim show differences in parasite growth inhibition with respect to their IC50 values [15, 18, 19]. In addition, parasites with a greater number of PfDHFR mutations showed increased IC50 values for pyrimethamine but much less of an increase in IC50 values for trimethoprim [19]. Trimethoprim is administered in more frequent doses and has a shorter half life than pyrimethamine, and thus exerts different (i.e., less) selection pressure on the parasite. Hence, it has been inferred that cross-resistance for trimethoprim and pyrimethamine is incomplete [15, 18, 19]. In fact, trimethoprim can eradicate the pyrimethamine-resistant parasite in vivo and in vitro [20, 21]

TS therapy was also observed to select for parasites with a lower number of PfDHFR mutations in Uganda, but not in Sudan or Mali [22–24]. There could be several reasons for the differences between our results and the results reported elsewhere [22, 23]. First, the combined use of SP and TS was probably more frequent in Car Nicobar during the period after the tsunami (January–March 2005; as mentioned above, >70% of patients with malaria received both TS and SP during this period) than in Sudan and Mali. Second, the number of PfDHFR-PfDHPS 2-locus mutations—and thus the level of SP resistance—was higher among the isolates collected prior to the tsunami in Car Nicobar than in the baseline isolates analyzed in these 2 countries [22, 23]. Therefore, because of the different clinical setting in Car Nicobar, trimethoprim and pyrimethamine could have had an antagonistic effect—whereas sulfadoxine and sulfamethoxazole had a synergistic effect—on selection for mutations of their respective target enzymes in the parasite population. Although the effects of SP and TS on these parasite enzymes have been studied separately, there is a lack of experimental data that examines the combined effect of SP and TS on the mutation pattern of these 2 genes [18]

A novel K540N mutation similar to that observed in some prokaryotes [11] was observed in several P. falciparum isolates from group C and in 1 isolate from group D, collected during October 2005 and February 2006, respectively. However, this K540N mutation was not detected in any of the isolates collected at other time points. We postulate here that selection pressure produced the population of parasites with this novel mutation because of the atypical SP-TS antifolate drug combination that was being used during January–March 2005. In the absence of this drug pressure, we observed no selection for parasites with this mutation in 2007–2008 or before October 2005. Nevertheless, this hypothesis requires the support of experimental evidence. Computer modeling of PfDHPS with this mutation showed the K540N mutation to have a binding capacity to sulfa drugs similar to that of its counterpart with the K540E mutation (figure 5). The results of the present study suggest that during natural disasters, care should be taken to avoid using 2 antifolate drugs (SP and TS) in combination, as they will expedite the selection of parasites with higher levels of sulfa drug resistance and novel mutations in the target gene

Acknowledgments

We are grateful to the Biotechnology Information System (BTIS) of the Department of Biotechnology at All India Institute of Medical Sciences and Mrs. Shalini Narang for preparing the manuscript. We are also grateful to Drs. J.S. Tyagi, H.K. Prasad, V. Udhayakumar, S.S. Chauhan, and S. Sinha for discussions and critical evaluation of the manuscript

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Potential conflicts of interest: none reported
Financial support: Indian Council of Medical Research (to M.K.; grants 75/23/2000/-ECDII to Y.D.S. and 63/128/2001-BMS to P.K.); Ministry of Science and Technology, Department of Biotechnology (grants BT/04/042/85 and BT/PR2428/Med/13/092/2001 to Y.D.S. and junior research fellowship to P.M.); Council for Scientific and Industrial Research (senior research fellowship to A.A. and V.L.)