Evaluation of pfmdr-1 Polymorphisms and Parasites’ Population Diversity in Children with Acute Uncomplicated Malaria 5 Years Post-Adoption of Artemisinin-Based Combination Therapies

Mutatons on pfmdr1 gene have been implicated in drug resistance to chloroquine and the partner drugs in artemisinin-based combination therapies (ACTs), hence the need to evaluate the impact of ACTs five years after its adoption in Nigeria on pfmdr1 polymorphisms and parasite diversity. Parasite genomic DNA was isolated from children below 5 years in Ibadan in 2010. Nested PCR followed by restriction fragment length polymorphism (RFLP) detected pfmdr1 Y86, F184 and Y1246 mutant alleles were present in 27%, 56% and 48% of the isolates respectively, while nested PCR evaluated polymorphic regions of MSP-1, MSP-2 and GLURP genes and monoclonal infections were observed in 81.6%, 51.6% and 5.6% with multiplicity of infection being 1.8, 2.0 and 2.4 respectively. This study showed a relative decline in the prevalence of Y86, F184 and Y1246 mutant alleles, but no significant change in the parasite population diversity of P.falciparum in children in Ibadan, Nigeria.

Genetic Features of P. falciparum Parasites Collected in 2012-2016 and Anti-Malaria Resistance Along China-Myanmar Border

Backgrounds The therapeutic efficacy studies (TES) of T(DHA-PIP) for uncomplicated P. falciparum patients were implemented during 2012-2016 along China (Yunnan province)-Myanmar border, which verified the high efficacy of DHA-PIP. The study focusing on the genetic features of falciparum parasites and basing on in vivo parasite clearance time (PCT) were carried out to explore if they had produced potential resistance to DHA and PIP at molecular level. Methods The genetic features were investigated based on K13 propeller genotypes, Copy numbers of genes pfPM2 and pfmdr1 and 9 microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The PCT 50s basing on different K13 genotypes, sites, periods and copy numbers were compared.Results In NW (North-West Yunnan province bordering with Myanmar) area, F446I prevalence was 58.96% (79/134). No significant different PCT 50s presented among 3 K13 groups (Chi-Square=2.35, P=0.31, df=2) classified by K13 genotypes within NW area, but all of them were significantly shorter than that in SW (South-West Yunnan province bordering with Myanmar) area (P=0.036, t=-2.11, df=174) where isolates only showed wild K13 genotype. For the copy numbers of pfmdr1 gene, 14.63% (18/123) and 62.5% (10/16) parasite isolates showed double copies (≥ 1.6) in NW and SW areas separately, but for those of pfM2 gene, no parasite isolates did. Between isolates with single and double copies of pfmdr1 gene, no different PCT 50 presented. According to the mean He values, the four K13 groups were arranged as ML group (Menglian County in SW), F446I group, Others (Non-F446I K13 mutation) group and W (wild K13 genotype) group from low to high. The mean Fst values between ML and W groups were significantly higher than other 2 K13 group-W pairs (Paired-T Test: t=-2.659，df=8，P=0.029; t=-4.966, df=8，P=0.001). Conclusions According to our study, P. falciparum isolates in NW area and SW area are very different in genetic features. Artemisinin partial resistance, inferred from genetic marker, F446I, had independently appeared and spread in NW area during 2012-2016. With global application of ACTs, this pattern of artemisinin resistance producing was worth more attention. Fortunately, PIP-resistant feature basing on genetic analysis showed negative results in both areas. So, DHA-PIP was still recommended in antimalarial treatment along China-Myanmar border based on molecular data, which was agreed with the conclusion drawn from in vivo data obtained with same falciparum isolates. Trial registration: ISRCTN, ISRCTN 11775446. Registered 13 April 2020 - Retrospectively registered, http://www.isrctn.com/ISRCTN11775446.

In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia

Background Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments. Objectives To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity. Methods In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine. Results Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7–18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1–11.8) for Pfk13 WT parasites and was 12.9 nM (9.5–20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5–20.5) versus 9.1 nM (7.9–10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene. Conclusions Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria.

Efficacy of Artemisinin Base Combination Therapy and Genetic Diversity of Plasmodium falciparum from Uncomplicated Ma-laria Falciparum Patient in District of Pesawaran, Province of Lampung, Indonesia

Background: Malaria is an infectious disease caused by Plasmodium sp., that still prevalence in some part of Indonesia. District of Pesawaran is one of malaria endemic area in the Province of Lampung. The purpose of this study was to evaluate the efficacy of the ACT treatment in the District of Pesawaran Province of Lampung, Indonesia from Dec 2012 to Jul 2013 and the genetic variation of the Plasmodium falciparum also studied. Methods: This study was observational analytic study of falciparum malaria patients treated with ACT and primaquine (DHP-PQ and AAQ-PQ) at Hanura Primary Health Centre (Puskesmas). DNA isolation was done with QIAmp DNA Mini Kit. Amplification of PfMDR1, MSP1, and MSP2 genes was done with appropriate forward and reverse primer and procedures optimized first. PCR Product of PfMDR1 gene was prepared for sequencing. Data analysis was done with MEGA 6 software. Results: The results of this research are DHP-PQ effectiveness was still wellness among falciparum malaria patients in District of Pesawaran, Province of Lampung, Indonesia. There is Single-nucleotide mutation of N86Y of PfMDR1 gene. The dominant alleles found are MAD20 and 3D7 alleles with Multiplicity of Infection (MOI) are low. Conclusion: Therapy of DHP-PQ as an antimalarial falciparum in Pesawaran District, Lampung, Indonesia is still good. The genetic variation found was the SNP on the N86Y PfMDR1 gene, with dominant allele MAD20 and 3D7.

Molecular investigation of the Pfmdr1 gene of Plasmodium falciparum isolates in Henan Province imported from Africa

Efficacious antimalarial drugs are important for malaria control and elimination, and continuous monitoring of their efficacy is essential. The prevalence and distribution of Pfmdr1 were evaluated in African migrant workers in Henan Province. Among 632 isolates, 13 haplotypes were identified, NYSND (39.87%, 252/632), YYSND (2.85%, 18/632), NFSND (31.01%, 196/632), NYSNY (0.47%, 3/632), YFSND (13.77%, 87/632), NFSNY (0.32%, 2/632), YYSNY (2.06%, 13/632), YFSNY (0.16%, 1/632), N/Y YSND (1.90%, 12/632), N Y/F SND (6.17%, 39/632), N/Y Y/F SND (0.47%, 3/632), YYSN D/Y (0.16%, 1/632) and N/Y FSND (0.79%, 5/632). The highest frequency of NYSND was observed in individuals from North Africa (63.64%, 7/11), followed by South Africa (61.33%, 111/181), Central Africa (33.33%, 56/168), West Africa (28.94%, 68/235) and East Africa (27.03%, 10/37) (χ2 = 54.605, P < 0.05). The highest frequency of NFSND was observed in East Africa (48.65%, 18/37), followed by West Africa (39.14%, 92/235), Central Africa (26.79%, 45/168), South Africa (22.65%, 41/181) and North Africa (9.09%, 1/11) (χ2 = 22.368 P < 0.05). The mutant prevalence of codons 86 and 184 decreased. These data may provide complementary information on antimalarial resistance that may be utilized in the development of a treatment regimen for Henan Province.