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Colombia has four main malaria transmission zones. In vivo efficacy studies carried out in these areas showed big differences in the response of Plasmodium falciparum to treatment with sulphadoxine-pyrimethamine. In addition, there is still insufficient information about the genetics of P. falciparum populations. The objective of this study was to determine the haplotypes in dhfr and dhps genes of P. falciparum circulating in two distinct endemic zones. Samples from patients with non-complicated P. falciparum malaria were collected: 135 from Tumaco and 206 from Tierralta. Alleles 108 and 51 of the dhfr gene, and 437 and 540 of the dhps gene were analyzed by PCR/enzymatic restriction, while alleles 59 and 164 (dhfr), and 581(dhps) by PCR/dot blot/hybridization. Five different haplotypes were found, of which the triple mutant 51I/C59/108N/I164/437G/K540/A581 was the most frequent (54.6%). In Tumaco, the parasites with wild haplotype predominated, while mutant parasites predominated in Tierralta. Another interesting finding is the presence of the C59R mutation in the dhfr gene in two samples, a mutation rarely found in South America. These data provide information about parasite population genetics and highlight the importance of starting a long term molecular surveillance program., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure., Objectives: To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba., Materials and Methods: Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism., Results: The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito., Conclusions: The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

Point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum can lead to an increasing resistance of P. falciparum to pyrimethamine/sulphadoxine. We examined the prevalence of these mutations in 36 samples from Colombia. Analysed by polymerase chain reaction (PCR) for infection with P. falciparum, 25 (69%) tested positive. These positive isolates were tested further for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and following mutation-specific restriction enzyme digestion. Gene mutations occurred in both the DHFR and DHPS gene of the Colombian isolates, suggesting that resistance to antifolate drugs exists or may develop soon in Colombia.

The capacity of Optimal to diagnose malaria was compared with the thick smear test in two representative samples, one with acute febrile syndrome (AFS) n = 107, and another diagnosed by thick smear test (AFS + M) n = 82. The samples were chosen from patients at the malaria diagnostic clinic in Turbo, Antioquia, Colombia, between June and August 2000. The study was designed to be descriptive, prospective, and cross-sectional. The two tests were applied simultaneously in the AFS group (parallel, double blind design), and in sequential form in the AFS + M group. The thick smear test was the standard test. Optimal tests were carried out according to the manufacturer's instructions. In the parallel design, Optimal showed, for Plasmodium falciparum, a sensitivity of 40% [95% CI: 18-67], a specificity of 98% (95% CI: 92-100) and positive and negative predictive values of 75% (95% CI: 36-96) and 91% (95% CI: 83-96%), respectively. For Plasmodium vivax, it showed a sensitivity of 97% (95% CI: 82-100), a specificity of 89% (95% CI: 80-95) and positive and negative predictive values of 79% (95% CI: 62-90) and 98% (95% CI: 91-100). With the sequential design, Optimal showed a sensitivity of 67% (95% CI: 52-79) and 97% (95% CI: 83-100) for P. falciparum and P. vivax, respectively. For P. falciparum, the sensitivity was directly proportional to the parasitemia, while the sensitivity for P. vivax was independent from the parasitemia. The diagnostic values and operative characteristics of the thick smear test surpassed the Optimal test in its sensitivity for P. falciparum; the specificities were similar. Both tests were nearly identical in their diagnostic capacity for P. vivax. These results recommend that the thick smear test be retained as a routine or reference test for malaria diagnosis, with Optimal used as an ancillary test.

n a study parallel to the present one, we conducted a genotype characterization of Plasmodium falciparum based on isolates of patients infected with malaria, who come from a small location in Colombia. The analysis involved extraction of DNA from hematological smears, amplification of the dihydrofolate reductase gene and the dihydropteroate synthase (DHPS) gene for each sample by PCR, and detection through mutation-specific primers nested PCR of mutations associated with resistance to pyrimethamine and sulfadoxine. Given the difficulty in quantifying the DNA extracts due to the type of sample and its heterogeneity, different volumes of the product of the first PCR were tested as template for the nested PCR. Surprisingly, for some samples, we found contradictory results between determinations, which differed only in the amount of template used. This prompted a more general concern that in a natural isolate, where the parasite population is heterogeneous, the nested PCR with mutation-specific primers technique can produce erroneous results that underestimate the complexity of the sample. To test this hypothesis, we designed experiments in this study using position 581 of the DHPS gene as an indicator system and constructed samples simulating the heterogeneity of natural samples. In effect, our data show that the results obtained in the nested PCR can be altered by the amount of template used in the reaction and, therefore, some heterogeneous samples might be classified mistakenly as homogeneous, simple mutant or simple wild type. These observations may explain, at least in part, contradictory results found in the literature. Our data also suggest the need for a more cautious approach to interpretation of the results of nested PCR assays with mutation-specific primers and their implications in the definition of resistance to the pyrimethamine-sulfadoxine combination., (Copyright 2001 Elsevier Science (USA).)

A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.

Several point mutations in the dihydrofolate reductase (DHFR) gene of Plasmodium falciparum have been correlated with in vitro anti-folate drug resistance of laboratory and field isolates. Furthermore, two different point mutations that generate amino acid substitutions at the same position of the enzyme have been observed in all the isolates studied to date. These point mutations change a serine (Ser-108) in the wild type to an asparagine (Asn-108 mutation) or to a threonine (Thr-108 mutation). Using the polymerase chain reaction (PCR), it is possible to identify isolates that present these mutations. We used a mutation-specific PCR to screen 71 samples from several geographic locations of Colombia for the Asn-108 mutation (pyrimethamine resistance). In this initial screening 53 of 71 yielded amplification product with the DHFR mutation-specific primers. We further analyzed the 18 samples that did not amplify using a mutation-specific nested PCR. Of those 18 samples, seven amplified with primers specific for the Thr-108 mutation (proguanil resistance), one with the wild type (Ser-108), and 10 did not amplify. Of these 10 samples, three were identified as P. falciparum using a species-specific diagnostic nested PCR base on sequences from the small ribosomal RNA subunit gene. Overall, 51.6% of the samples amplified for the Asn-108 mutation, 10.9% for the Thr-108 mutation, 35.9% with the wild type specific primer, and 4.8% did not amplify with any of the DHFR primers. We observed variability in the frequency of the mutation between the different geographic location. The frequency of the Asn-108 and Thr-108 mutations in the state of Narifio was 25% each, while in Valle del Cauca the frequencies were 59% and 11%, respectively. These results contrast with observations in Brazil in which the Asn-108 mutation was found in 90% of the blood samples screened.