Sustained High Cure Rate of Artemether-Lumefantrine against Uncomplicated Plasmodium falciparum Malaria after 8 Years of Its Wide-Scale Use in Bagamoyo District, Tanzania.

We assessed the temporal trend of artemether-lumefantrine (AL) cure rate after 8 years of its wide-scale use for treatment of uncomplicated Plasmodium falciparum malaria from 2006 to 2014 in Bagamoyo district, Tanzania. Trend analysis was performed for four studies conducted in 2006, 2007-2008, 2012-2013, and 2014. Patients with acute uncomplicated P. falciparum malaria were enrolled, treated with standard AL regimen and followed-up for 3 (2006), 28 (2014), 42 (2012-2013), or 56 (2007-2008) days for clinical and laboratory evaluation. Primary outcome was day 28 polymerase chain reaction (PCR)-adjusted cure rate across years from 2007 to 2014. Parasite clearance was slower for the 2006 and 2007-2008 cohorts with less than 50% of patients cleared of parasitemia on day 1, but was rapid for the 2012-2013 and 2014 cohorts. Day 28 PCR-adjusted cure rate was 168/170 (98.8%) (95% confidence interval [CI], 97.2-100), 122/127 (96.1%) (95% CI, 92.6-99.5), and 206/207 (99.5%) (95% CI, 98.6-100) in 2007-2008, 2012-2013, and 2014, respectively. There was no significant change in the trend of cure rate between 2007 and 2014 (χ2trend test = 0.06, P = 0.90). Pretreatment P. falciparum multidrug-resistant gene 1 (Pfmdr1) N86 prevalence increased significantly across years from 13/48 (27.1%) in 2006 to 183/213 (85.9%) in 2014 (P < 0.001), and P. falciparum chloroquine resistance transporter gene (Pfcrt) K76 prevalence increased significantly from 24/47 (51.1%) in 2006 to 198/205 (96.6%) in 2014 (P < 0.001). The AL cure rate remained high after 8 years of its wide-scale use in Bagamoyo district for the treatment of uncomplicated P. falciparum malaria despite an increase in prevalence of pretreatment Pfmdr1 N86 and Pfcrt K76 between 2006 and 2014.

pfmdr1 (Plasmodium falciparum multidrug drug resistance gene 1): a pivotal factor in malaria resistance to artemisinin combination therapies.

INTRODUCTION: Plasmodium falciparum, the deadly agent of malaria, is notorious for its capacity to develop drug resistance. Treatment failures of artemisinin therapy regimens (ACTc), the present mainstay, is emerging. The transporter coding pfmdr1 gene is a central node in this process, having been associated with in vitro and in vivo parasite response to a broad range of ACT antimalarials. Areas covered: The review covers the historical origins of the pfmdr1 discovery, followed by a detailed description of its sequence and expression characteristics, as well as the structural and functional characteristics of its coded transmembrane protein. pfmdr1 association with ACT drugs response in vivo and in vitro is thoroughly reviewed. A reference is made to significant compounds presently in the development pipeline. The literature search was focused on Pubmed based searches with occasional resource to edited books, World Health Organization documentation and conference reports for adding valuable details. Expert commentary: Pfmdr1 has emerged as the central gene in P. falciparum ACT resistance. Understanding the basis of this role is critical for epidemiologic surveillance and design of improved resistance-refractory antimalarials. Specifically, unveiling situations of drug collateral sensitivity associated with specific pfmdr1 genetic variation will provide opportunities for personalized optimal therapy options.

Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 µL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt.

Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment in Tanzanian children with acute uncomplicated malaria.

BACKGROUND: This study aimed to explore Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment with artemisinin-based combination therapy in children with clinical malaria in a high transmission area in Africa. METHODS: A total of 50 children aged 1-10 years with acute uncomplicated P. falciparum malaria in Bagamoyo District, Tanzania, were enrolled. Participants were hospitalized and received supervised standard treatment with artemether-lumefantrine according to body weight in six doses over 3 days. Blood samples were collected 11 times, i.e. at time of diagnosis (-2 h) and 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 h after initiation of treatment. Parasite population dynamics were assessed using nested polymerase chain reaction (PCR)-genotyping of merozoite surface protein (msp) 1 and 2. RESULTS: PCR-analyses from nine sequential blood samples collected after initiation of treatment identified 20 and 21 additional genotypes in 15/50 (30%) and 14/50 (28%) children with msp1 and msp2, respectively, non-detectable in the pre-treatment samples (-2 and 0 h combined). Some 15/20 (75%) and 14/21 (67%) of these genotypes were identified within 24 h, whereas 17/20 (85%) and 19/21 (90%) within 48 h for msp1 and msp2, respectively. The genotype profile was diverse, and varied considerably over time both within and between patients, molecular markers and their respective families. CONCLUSION: PCR analyses from multiple blood samples collected during the early treatment phase revealed a complex picture of parasite sub-populations. This underlines the importance of interpreting PCR-outcomes with caution and suggests that the present use of PCR-adjustment from paired blood samples in anti-malarial drug trials may overestimate assessment of drug efficacy in high transmission areas in Africa.The study is registered at http://www.clinicaltrials.gov with identifier NCT00336375.

Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in Africa.

Plasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.

Polymorphism in PfMRP1 (Plasmodium falciparum multidrug resistance protein 1) amino acid 1466 associated with resistance to sulfadoxine-pyrimethamine treatment.

Sulfadoxine-pyrimethamine (SP) remains widely recommended for intermittent preventive treatment against Plasmodium falciparum malaria for pregnant women and infants in Africa. Resistance to SP is increasing and associated primarily with mutations in the P. falciparum dhfr (Pfdhfr) and Pfdhps genes. This study aimed to explore the hypothetical association of genetic alterations in the P. falciparum multidrug resistance protein gene (Pfmrp1) with the in vivo response to SP by detecting the selection of single nucleotide polymorphisms (SNPs) following standard single-dose treatment administered to children with acute uncomplicated P. falciparum malaria in Tanzania. We detected significant selection of parasites carrying the Pfmrp1 1466K allele in samples from children with recrudescent infections, with 12 (100%) of 12 such samples being positive for this allele, compared to 52 (67.5%) of 77 baseline samples (P = 0.017), in parallel with the selection of the Pfdhfr Pfdhps quintuple mutant haplotype in cases of recrudescence (P = 0.001). There was no association between the 1466K SNP and the Pfdhfr Pfdhps quintuple mutation, indicating independent selections. Our data point for the first time to a role for a P. falciparum multidrug resistance protein homologue in the antimalarial activity of SP. Moreover, they add to the growing evidence of the potential importance of Pfmrp1 in antimalarial drug resistance.

CYP2C8 polymorphism among the Portuguese.

Cytochrome P450 2C8 (CYP2C8) is a polymorphic phase I drug-metabolising enzyme involved in the metabolism of a wide variety of xenobiotics, as well as a proposed player in the regulation of vascular tone. Polymorphisms in this gene may have an impact on the metabolism of therapeutic drugs such as paclitaxel and verapamil. In this report we have determined the frequencies of the main non-synonymous CYP2C8 alleles, 805A>T (CYP2C8*2), 416G>A/1196A>G (CYP2C8*3) and 792C>G (CYP2C8*4) in a sample representative of Portuguese Caucasians. The allelic frequencies determined were 1.2%, 19.8%, and 6.4% for CYP2C8*2, CYP2C8*3, and CYP2C*4, respectively. The observed CYP2C8*3 prevalence is significantly different from the frequencies previously reported in North European populations.

Efficacy of artesunate plus amodiaquine versus that of artemether-lumefantrine for the treatment of uncomplicated childhood Plasmodium falciparum malaria in Zanzibar, Tanzania.

BACKGROUND: This is the first clinical trial comparing the efficacy of artesunate plus amodiaquine (ASAQ) and artemether-lumefantrine (AL)--the major artemisinin-based combination therapy (ACT) candidates for treatment of malaria in Africa--that involved an extended, 42-day follow-up period, polymerase chain reaction-adjusted parasitological cure rates (PCR APCRs), and systematic analyses of genetic markers related to quinoline resistance. METHODS. A total of 408 children with uncomplicated Plasmodium falciparum malaria in Zanzibar, Tanzania, were enrolled. Children who were 6-8 months of age and/or who weighed 6-8 kg were assigned to receive ASAQ for 3 days. Children who were 9-59 months of age and who weighted > or =9 kg were randomly assigned to receive either ASAQ or AL for 3 days in standard doses. Intention-to-treat analyses were performed. RESULTS: Age- and weight-adjusted PCR-APCRs by follow-up day 42 were 91% (188 of 206 patients) in the ASAQ group and 94% (185 of 197 patients) in the AL group (odds ratio [OR] for the likelihood of cure, 2.07; 95% confidence interval [CI], 0.84-5.10; P=.115). A total of 5 and 7 recrudescences occurred after day 28 in the ASAQ and AL groups, respectively. On the assumption that 10 malaria episodes with uncertain PCR results were recrudescences, PCR-APCRs decreased to 88% in the ASAQ group and to 92% in the AL group. Unadjusted cure rates by day 42 were 56% (116 of 206 patients) in the ASAQ group versus 77% (151 of 197 patients) in the AL group (OR, 2.55; 95% CI, 1.66-3.91; P<.001). Rates of reinfection by day 42 were 36% (65 of 181 patients) in the ASAQ arm versus 17% (31 of 182 patients) in the AL arm (OR, 0.37; 95% CI, 0.22-0.60; P<.001). A significant selection of P. falciparum multidrug resistance gene 1 allele 86N was found in isolates associated with reinfection after AL treatment, compared with isolates at baseline (2.2-fold increase; P<.001). CONCLUSIONS: Both treatments were highly efficacious, but AL provided stronger prevention against reinfection. The high proportion of recrudescences found after day 28 and the genetic selection by the long-acting partner drug underlines the importance of long follow-up periods in clinical trials. A long follow-up duration and performance of PCR genotyping should be implemented in programmatic surveillance of antimalarial drugs.

Short report: Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum.

The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive, and cost-effective DNA extraction methods. We describe a new Tris-EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The method was evaluated against the commonly used methanol and Chelex methods, regarding polymerase chain reaction detection of Plasmodium falciparum parasites from samples stored for 1-2 years. The sensitivity of detection was dependent on the parasite density and type of filter paper. For 3MM Whatman filter paper, the sensitivity was 100%, 73%, and 93% for the TE, methanol, and Chelex methods, respectively. For the longer stored 903 Schleicher & Schuell filter paper, the sensitivity was 93%, 73%, and 0%, respectively. This rapid, simple, and inexpensive extraction method generated superior results from archived specimens compared with the two standard methods and may represent a useful tool in molecular epidemiologic studies.

CYP3A4 and MDR1 alleles in a Portuguese population.

Polymorphisms in cytochrome P450 CYP3A4 and multidrug resistance (MDR) 1 genes coding for the important drug-metabolising CYP3A4 and the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) are poorly documented in the Portuguese population. In this study we have determined the frequencies of CYP3A4 and MDR1 alleles in Portuguese Caucasians. Both genes were simultaneously analysed as these genes are known to be frequently co-induced and their products to show a pronounced overlap of substrates. CYP3A4 A-392G (CYP3A4*1B), T673C (CYP3A4*2) and MDR1 T-129C, G2677T and C3435T single nucleotide polymorphisms (SNPs) were analysed in 100 individuals from the southern region of the country. We observed a frequency of 4.0% for CYP3A4*1B, not significantly different from that reported on other Caucasian European populations. CYP3A4*2 was found at an allele frequency of 4.5%, constituting the first report of the presence of this allele outside the Finnish population. Significant differences were found concerning the MDR1 C3435T SNP frequency (64.5%) compared with other European populations, while no differences were found concerning G2677T (47.5%) or T-129C (5%) SNPs. Linkage between the C3435T and G2677T SNPs was observed, although not as evidently as documented in other Caucasian populations. No preferential associations were detected between CYP3A4 and MDR1 alleles.

CYP3A4*1B and NAT2*14 alleles in a native African population.

Single nucleotide polymorphisms were examined in the cytochrome 450 3A4 (CYP3A4) and N-acetyltransferase 2 (NAT2) genes, which code for major mediators of the metabolism of a wide variety of therapeutic drugs, as well as xenobiotics. We determined, in a population from Guinea-Bissau, the frequencies of CYP3A4 and NAT2 variants expected to be prevalent among Africans, due to the high frequency previously observed in African Americans. The observed frequencies were 72% for CYP3A4*1B and 19.2% for the NAT2 191 G>A variant. The high frequency found for these potentially function-altering polymorphisms suggests the possibility of impaired metabolism through CYP3A4 and NAT2 in this population. Strikingly, the frequency observed for the NAT2 191 G>A single nucleotide polymorphism (SNP), associated with the slow acetylator phenotype, was significantly higher than found in other African populations, suggesting the existence of a west to east gradient across Sub-Saharan Africa. The prevalence of these variants may be relevant with regard to therapeutic efficacy in African populations for it may potentially affect drug clearance and consequently, increase the incidence of side effects and drug-drug interactions.