Quinine Susceptibility Research Articles

Prevalence of mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, and association with ex vivo susceptibility to common anti-malarial drugs against African Plasmodium falciparum isolates

BackgroundThe Plasmodium falciparum chloroquine transporter gene (pfcrt) is known to be involved in chloroquine and amodiaquine resistance, and more particularly the mutations on the loci 72 to 76 localized within the second exon. Additionally, new mutations (T93S, H97Y, C101F, F145I, M343L, C350R and G353V) were recently shown to be associated with in vitro reduced susceptibility to piperaquine in Asian or South American P. falciparum strains. However, very few data are available on the prevalence of these mutations and their effect on parasite susceptibility to anti-malarial drugs, and more particularly piperaquine in Africa.MethodsA molecular investigation of these mutations was performed in 602 African P. falciparum parasites collected between 2017 and 2018 on malaria patients hospitalized in France after a travel in African countries. Associations between genotypes and in vitro susceptibilities to piperaquine and standard antimalarial drugs were assessed.ResultsNone of the mutations, previously described as associated with piperaquine resistance, was found in the 602 P. falciparum African isolates. The K76T mutation is associated with resistance to chloroquine (p < 0.0002) and desethylamodiaquine (p < 0.002) in Africa. The K76T mutation is not associated with in vitro reduced susceptibility to piperaquine. The mutation I356T, identified in 54.7% (n = 326) of the African isolates, was significantly associated with reduced susceptibility to quinine (p < 0.02) and increased susceptibility to mefloquine (p < 0.04). The K76T and I356T mutations were significantly associated in West African isolates (p = 0.008).ConclusionNone of the mutations in pfcrt found to be associated with piperaquine reduced susceptibility in Asia or South America (T93S, H97Y, C101F, F145I, M343L C350R and G353V) were found in the 602 African isolates including the three isolates with reduced susceptibility to piperaquine. The K76T mutation, involved in resistance to chloroquine and amodiaquine, and the I356T mutation were not associated with in vitro reduced susceptibility to piperaquine. Differences in mefloquine susceptibility between I356 and 356T isolates were, while statistically different, minimal. Further analyses are needed with a more important sample size from the same geographic area to confirm the role of the I356T mutation on quinine susceptibility.

Prevalence of Plasmodium falciparum anti-malarial resistance-associated polymorphisms in pfcrt, pfmdr1 and pfnhe1 in Muheza, Tanzania, prior to introduction of artemisinin combination therapy.

BackgroundA report of the chloroquine and amodiaquine resistance pfcrt-SVMNT haplotype in Tanzania raises concern about high-level resistance to the artesunate-amodiaquine combination treatment widely employed in Africa. Mutations in the pfmdr1 multi-drug resistance gene may also be associated with resistance, and a highly polymorphic microsatellite (ms-4760) of the pfnhe1 gene involved in quinine susceptibility has not been surveyed in Tanzania.MethodsA total of 234 samples collected between 2003 – 2006 from an observational birth cohort of young children in Muheza, Tanzania were analysed. In these children, 141 cases of P. falciparum infections were treated with AQ and 93 episodes were treated with QN. Haplotypes of pfcrt and pfmdr1 were determined by a Taqman assay, and ms-4760 repeats in pfnhe1 were assessed by nested PCR amplification and direct sequencing. Parasite population diversity was evaluated using microsatellite markers on five different chromosomes.ResultsThe pfcrt-CVIET haplotype was present alone in 93.6% (219/234) of the samples over the study period; the wild-type chloroquine- and amodiaquine-sensitive haplotype pfcrt-CVMNK was present in 4.3% (10/234) of the samples; and both haplotypes were present in 2.1% (5/234) of the samples. No significant change in wild-type pfcrt-CVMNK prevalence was evident over the 4-year period of the study. The pfcrt-SVMNT haplotype associated with high-level amodiaquine resistance was not detected in this study. The pfmdr1 locus was genotyped in 178 of these samples. The pfmdr1-YYNY haplotype predominated in 67.4% (120/178) of infections and was significantly associated with the pfcrt-CVIET haplotype. All samples carried the wild-type pfmdr1-N1042 codon. The ms-4760 repeat on pfnhe1 locus displayed 12 distinct haplotypes with ms-4760-1 predominating in the population. Analysis of these haplotypes showed no association of a particular haplotype with quinine treatment outcome.ConclusionThe pfcrt-CVIET chloroquine resistance haplotype dominated in the collection of P. falciparum samples from Muheza. The pfcrt-SVMNT haplotype, which threatens the efficacy of amodiaquine and was reported in the same time period from Korogwe, Tanzania, 40 Km from Muheza, was not detected. Relative low prevalence of pfcrt-SVMNT in Africa may result from genetic or other factors rendering P. falciparum less supportive of this haplotype than in South America or other regions.Trial registrationTrial Protocol Number: 08-I-N064.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0642-2) contains supplementary material, which is available to authorized users.

Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 genes are associated with reduced in vitro activities of quinine in Plasmodium falciparum isolates from western Kenya.

In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pfmdr1codons 86 and 184, Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC50), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and quinine susceptibility (P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pfmdr1-N86, Pfmdr1-Y184, and Pfcrt-K76, respectively). In addition, quinine IC50 values for parasites with Pfnhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats (P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger transporter (Pfnhe-1) gene in 393 isolates from Dakar, Senegal

BackgroundAlthough the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility.MethodsIn this context, to further analyse associations between Pfnhe-1 ms4760 polymorphisms and QN susceptibility, 393 isolates freshly collected between October 2009 and January 2010 and July 2010 and February 2011, respectively, at the Hôpital Principal de Dakar, Senegal were assessed ex vivo for QN susceptibility, and their genes were amplified and sequenced.ResultsOf the 393 Plasmodium falciparum clinical isolates collected, 145 were successfully cultured. The 145 QN IC50s ranged from 2.1 to 1291 nM, and 17 isolates (11.7%) exceed the QN reduced susceptibility threshold of 611 nM. Among the 393 P. falciparum clinical isolates, 47 different alleles were observed. The three most prevalent profiles were ms4760-1 (no = 72; 18.3%), ms4760-3 (no = 65; 16.5%) and ms4760-7 (no = 40; 10.2%). There were no significant associations observed between QN IC50 values and i) the number of repeats of DNNND in block II (p = 0.0955, Kruskal-Wallis test); ii) the number of repeats of DDNHNDNHNND in block V (p = 0.1455, Kruskal-Wallis test); or iii) ms4760 profiles (p = 0.1809, Kruskal-Wallis test).ConclusionsPfnhe-1 ms4760 was highly diverse in parasite isolates from Dakar (47 different profiles). Three profiles (ms4760-1, ms4760-3 and ms4760-7) were predominant. The number of repeats for block II (DNNND) or block V (DDNHNDNHNND) was not significantly associated with QN susceptibility. New studies, and especially in vivo studies, are necessary to confirm the role of Pfnhe-1 ms4760 as a marker of QN resistance.

Differential Association of Plasmodium falciparum Na + /H + Exchanger Polymorphism and Quinine Responses in Field- and Culture-Adapted Isolates of Plasmodium falciparum

Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na(+)/H(+) exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC(50)) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.

Polymorphism of Plasmodium falciparum Na+/H+ exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam

BackgroundThe Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam.MethodNinety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay.ResultsSignificant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7.ConclusionsThe haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.

Multidrug-Resistant Genotypes ofPlasmodium falciparum, Myanmar

We performed a molecular epidemiologic survey of mutations associated with drug-resistance genes in Plasmodium falciparum in northeastern Myanmar. In this region, 3 highly mutated drug-resistance haplotypes and 1 associated with decreased quinine susceptibility were prevalent, which suggests that parasites may be resistant to multiple commonly used antimalarial drugs.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

BackgroundQuinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa.MethodsThe sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo.ResultsPolymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility.ConclusionsThe polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure.

Plasmodium falciparumNa+/H+Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine

Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC(50)s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC(50) of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC(50) of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC(50) of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC(50) of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC(50) of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC(50) of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.

Dissecting the components of quinine accumulation in Plasmodium falciparum

Although quinine, the active ingredient of chinchona bark, has been used in the treatment of malaria for several centuries, there is little information regarding the interactions of this drug with the human malaria parasite Plasmodium falciparum. To better understand quinine's mode of action and the mechanism underpinning reduced responsiveness, we have investigated the factors that contribute to quinine accumulation by parasites that differ in their susceptibility to quinine. Interestingly, passive distribution, in accordance with the intracellular pH gradients, and intracellular binding could account for only a small fraction of the high amount of quinine accumulated by the parasites investigated. The results of trans-stimulation kinetics suggest that high accumulation of quinine is brought about by a carrier-mediated import system. This import system seems to be weakened in parasites with reduced quinine susceptibility. Other data show that polymorphisms within PfCRT are causatively linked with an increased verapamil-sensitive quinine efflux that, depending on the genetic background, resulted in reduced quinine accumulation. The polymorphisms within PfMDR1 investigated did not affect quinine accumulation. Our data are consistent with the model that several factors, including acidotropic trapping, binding to intracellular sites and carrier-mediated import and export transport systems, contribute to steady-state intracellular quinine accumulation.