We evaluated associations between key polymorphisms in target genes and responses to treatment with sulfadoxine-pyrimethamine (SP) or amodiaquine (AQ) for uncomplicated Plasmodium falciparum malaria in Bobo-Dioulasso, Burkina Faso. Presence of the dihydrofolate reductase (dhfr) 108N or 59R mutations (but not dhfr 51I or dihydropteroate synthetase [dhps] 437G) and P. falciparum chloroquine resistance transporter (pfcrt) 76T or P. falciparum multidrug resistance 1 (pfmdr1) 86Y or 1246Y mutations (but not pfmdr1 184F) predicted recrudescence after treatment with SP and AQ, respectively. Treatment led to significant increases in the prevalence of the same mutations (except 1246Y) in new infections that presented after therapy. The dhfr 164L and dhps 540E mutations were not seen in any isolates. These results clarify the key roles of a small number of mutations in P. falciparum resistance to SP and AQ in west Africa.
"The persistent treatment of P. falciparum infections with CQ will subsequently encourage the selection of CQ resistant strains [20-23]. The danger is that there could be subsequent selection of additional changes in genes regulating P. falciparum response to CQ [24-28]. Such a situation will favour cross-resistance between CQ and amodiaquine, which is co-partner of artesunate, the first ACT deployed in Ghana [28-30]. "
[Show abstract][Hide abstract] ABSTRACT: Background
After years of disuse of chloroquine (CQ) as first-line anti-malarial drug in Ghana, reports from molecular studies conducted in parts of the country indicate varying prevalence of T76 mutation in the pfcrt gene. This situation has several health implications, one being that mutations that confer resistance to CQ have been reported to show substantial cross-resistance to other anti-malarial drugs. It is important to identify some of the factors contributing to the continuous presence of CQ resistance markers in the country. This study determined the prevalence of T76 mutation in pfcrt gene of Plasmodium falciparum isolates collected from selected areas of the Central region of Ghana and correlated with the level of CQ use in these areas.
Plasmodium falciparum DNA was extracted from collected blood-blot filter paper samples in the study sites. The prevalence of T76 point mutation in pfcrt gene was assessed using nested PCR followed by RFLP. CQ from pharmacy and chemical shops was obtained using mystery buying method. The extent of CQ use by the participants was determined by measuring the level of the drug in their urine samples using the Saker-Solomon method.
Of the 214 P. falciparum isolates analysed, 71.9% were found to have T76 mutation of pfcrt gene. The study revealed that 14.49% of community pharmacies and chemical shops had stocks of CQ for sale while 16.9% of the participants had CQ in their urine samples. There is five times more risks of becoming infected with CQ resistant strain for staying in an area where CQ is stocked for sale [RR = 0.20, p < 0.0001] and thirteen times more risks of having CQ-resistant mutant from those who still use CQ than non-users [OR = 0.08, p < 0.0001].
This study has shown that high variation in the prevalence of T76 mutations of P. falciparum is linked with the level of CQ stocking and usage within study area.
"Polymorphisms in another gene, pfmdr1, which encodes a protein homologous to transporters that mediate drug resistance in other organisms, modulate levels of sensitivity to multiple drugs . In Africa, the pfmdr1 N86Y, Y184F and D1246Y polymorphisms are common, and the 86Y and 1246Y mutations are associated with decreased sensitivity to chloroquine and amodiaquine [4-6]. Interestingly, wild type sequences at these same alleles lead to decreased sensitivity to artemisinins and the ACT partner drugs lumefantrine and mefloquine [5,7,8]. "
[Show abstract][Hide abstract] ABSTRACT: Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.
For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.
SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K = 0.77) for pfcrt K76T, 79% (K = 0.55) for pfmdr1 N86Y, 83% (K = 0.65) for pfmdr1 Y184F, and 91% (K = 0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.
The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.
"In contrast, the parasite populations in western Africa seem to be mostly sensitive to SP
[7,14-16], and IPTp-SP has proven to be highly effective and efficacious in clinical trials and observational studies
[5,15]. However, spread of SP resistance from eastern and southern Africa, or the de novo development of high-level SP resistance may occur and monitoring of the effectiveness of SP when employed as IPTp is essential. "
[Show abstract][Hide abstract] ABSTRACT: Intermittent Preventive Treatment in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) is widely used for the control of malaria in pregnancy in Africa. The emergence of resistance to SP is a concern requiring monitoring the effectiveness of SP for IPTp.
This was an in-vivo efficacy study to determine the parasitological treatment response and the duration of post-treatment prophylaxis among asymptomatic pregnant women receiving SP as part of IPTp in Mali and Burkina-Faso. The primary outcome was the PCR-unadjusted % of patients with parasites recurrence by day 42 defined as a positive diagnostic test by malaria smear at any visit between days 4 and 42. Treatment failure was based on the standard World Health Organization criteria. The therapeutic response was estimated using the Kaplan-Meier curve.
A total of 580 women were enrolled in Mali (N=268) and Burkina-Faso (N=312) and followed weekly for 42days. Among these, 94.3% completed the follow-up. The PCR-unadjusted cumulative risk of recurrence by day 42 was 4.9% overall, and 3.2% and 6.5% in Mali and Burkina Faso respectively (Hazard Ratio [HR] =2.14, 95%, CI [0.93-4.90]; P=0.070), and higher among the primi- and secundigravida (6.4%) than multigravida (2.2%, HR=3.01 [1.04-8.69]; P=0.042). The PCR-adjusted failure risk was 1.1% overall (Mali 0.8%, Burkina-Faso 1.4%). The frequencies (95% CI) of the dhfr double and triple mutant and dhps 437 and 540 alleles mutant genotype at enrolment were 24.2% (23.7-25.0), 4.7% (4.4-5.0), and 21.4% (20.8-22.0) and 0.37% (0.29-0.44) in Mali, and 7.1% (6.5-7.7), 44.9% (43.8-46.0) and 75.3% (74.5-76.2) and 0% in Burkina-Faso, respectively. There were no dhfr 164L or dhps 581G mutations.
SP remains effective at clearing existing infections when provided as IPTp to asymptomatic pregnant women in Mali and Burkina. Continued monitoring of IPTp-SP effectiveness, including of the impact on birth parameters in this region is essential.
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