Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites ...in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections—S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30–80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples—S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2–12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent—S108T (90%), with A16V and the 108T+16V double mutant (49–57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.
In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, ...sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha.
A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion.
Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent.
This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll.
Plasmodium falciparum genotyping with molecular polymorphic markers is widely employed to distinguish recrudescence from re-infection in antimalarial drug efficacy monitoring programmes. However, ...limitations occur on agarose gel DNA measurements used to resolve the polymorphisms. Without empirical data, the current distinction of pre- and post-treatment bands, as persistent or new infection, is subjective and often varying by author. This study measures empirical tolerance limits for classifying different-sized bands as same or different alleles during MSP2 genotyping.
P. falciparum field samples from 161 volunteers were genotyped by nested PCR using polymorphic MSP2 family-specific primers. Data were analysed to determine variability of band size measurements between identical MSP2 alleles randomized into different agarose lanes.
The mean (95% CI) paired difference in band size between identical alleles was 9.8 bp (1.48 - 18.16 bp, p = 0.022) for 3D7/IC and 2.54 (-3.04 - 8.05 bp, p = 0.362) for FC27. Based on these findings, pre- and post-treatment samples with 3D7/IC alleles showing less than 18 bp difference corresponded to recrudescence, with 95% confidence, while greater difference indicated new infection. FC27 allele differences were much narrower. For both 3D7/IC and FC27 amplicon, allele detection sensitivity was significantly higher with 13 mul compared to 20 mul or 30 mul lane loading volumes.
During MSP genotyping, it is useful to standardize classifications against measurement of background variability on identical alleles, in order to obtain reliable findings. It is critical to use a fixed optimal lane loading volume for constant allele patency, to avoid the disappearance or false appearance of new infection.
Rice grain quality is an important factor that has a great influence on its market value and consumer acceptance. It is determined by three parameters controlling the cooking and eating qualities of ...rice (amylose content, gelatinization temperature and gel consistency) and by the aroma, which becomes a criterion increasingly preferred by consumers. Molecular characterization of specific genomic regions of rice genotypes by trait specific markers can help in the development of suitable breeding program. This study was conducted at AfricaRice Regional station, Saint-Louis, Senegal. 30 rice genotypes commonly used in Africa were evaluated using eight simple sequence repeat (SSR) markers linked to the cooking, eating properties, and the aroma. The total number of alleles was 45 with an average of 5.63 allele per locus. The number of alleles per marker varied from three for RM204 to eight for RM190 and RM342A and the effective number of alleles varied from 1.66 for RM204 to 6.16 for RM342A. The polymorphic information content (PIC) varied from 0.39 to 0.83 and the allele frequency ranged from 0.015 to 0.75. A maximum genetic similarity of 1 was observed between Gambiaka Kokoum and Gambiaka Burkina Faso, Basmati 270 and Basmati 370, Sahel 108 and Sahel 201, Sahel 108 and Sahel 208, Sahel 201 and Sahel 208, Sahel 202 and Sahel 209, and Sahel 305 and Sahel 317. The Sahel varieties found with maximum genetic similarity have the same amylose content, but different gelatinization temperature except Sahel 305 and Sahel 317 which have the same cooking and eating properties. Therefore, more markers are needed to discriminate those varieties. Minimum genetic similarity was observed between traditional aromatic rice Basmati 370 and the landrace Gambiaka Nigeria. The unweighted pair-groups method using arithmetic averages (UPGMA) cluster analysis of these cultivars enabled the classification of our varieties in five major groups with additional sub-clusters in groups 2, 3 and 4. Groups 1 and 2 composed of aromatics varieties, group 3 gathered the three improved Sahel aromatic varieties, group 4 was the most diversified group with three sub-clusters and group 5 corresponded to the traditional varieties Gambiaka. The results of this study indicated that the use of trait specific SSR markers enabled to group the varieties according to their cooking and eating quality and the aroma and therefore can be very useful in breeding rice varieties harboring good cooking and eating quality traits and aroma in rice breeding program.
PDF
