Gisela Henriques

Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated with ACT.

Background The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42. Methods DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure. Results Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72–76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia. Conclusions Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence.

First survey for Babesia bovis and Babesia bigemina infection in cattle from Central and Southern regions of Portugal using serological and DNA detection methods.

Incidence of bovine babesiosis in Portugal is currently unknown. In this study, a first survey of Babesia bovis and Babesia bigemina infection in cattle was carried out using blood samples from 406 clinically healthy individuals from different districts from Central and Southern regions of Portugal and analyzed by indirect enzyme linked immunosorbent assay (iELISA) and nested polymerase chain reaction (nPCR). Overall, serological testing revealed that 79% and 52% of cattle were positive for B. bovis and B. bigemina antibodies, respectively, whereas nPCR testing detected 71% and 34% cattle infected with B. bovis and B. bigemina protozoan, respectively. This is the first report of the prevalence of B. bovis and B. bigemina in cattle obtained by serological and DNA analysis studies in Central and Southern regions of Portugal. These data suggests high incidence of Babesia sp. infection in Portugal and can be used for designing adequate control programs.

Artemisinin resistance in rodent malaria - mutation in the AP2 adaptor μ-chain suggests involvement of endocytosis and membrane protein trafficking

BackgroundThe control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi.MethodsHere, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared.ResultsAn increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the μ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene.ConclusionAn increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.

Experimental Evolution of Resistance to Artemisinin Combination Therapy Results in Amplification of the mdr1 Gene in a Rodent Malaria Parasite

Background Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. Methodology/Principal Findings We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN) + mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN + MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN + MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. Conclusion Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as yet unidentified, are likely to be involved.

Lack of K13 mutations in Plasmodium falciparum persisting after artemisinin combination therapy treatment of Kenyan children

AbstractBackgroundStudies in Southeast Asia reported a strong relationship between polymorphisms at the propeller domain of the Kelch 13 (K13) protein encoded by the Plasmodiumfalciparumk13(pfk13) gene and delayed parasite clearance after artemisinin treatment. In Africa, P. falciparum remains susceptible and combination therapy regimens which include an artemisinin component display good efficacy. Using quantitative real-time PCR (qPCR), sub-microscopic persistence of P. falciparum has previously been reported in one-third of children treated with artemisinin combination therapy (ACT) in western Kenya. In this study, further investigation was made to evaluate whether these sub-microscopic residual parasites also harbour mutations at the propeller region of pfk13 and whether the mutations, if any, affect treatment outcome.MethodsThe pfk13 propeller domain was genotyped in DNA samples obtained in 2009 from Kenyan children treated with artemether–lumefantrine (AL) and dihydroartemisinin–piperaquine (DP). Paired samples at pre-treatment (day 0) and day of treatment failure (day 28 or 42) for 32 patients with documented recurrent parasitaemia were available for genotyping. Additional day 3 DNA samples were available for 10 patients.ResultsNo mutation associated with artemisinin resistance in Southeast Asia was observed. Only one DP-treated patient harboured a non-synonymous mutation at codon 578 (A578S) of pfk13-propeller gene in the day 0 sample, but this allele was replaced by the wild-type (A578) form on day 3 and on the day of recurrent parasitaemia. The mutation at amino acid codon 578 showed no association with any phenotype. Polymorphisms in pfk13 were not responsible for parasite persistence and gametocyte carriage in the children treated with ACT.ConclusionThis study contributes to the ongoing surveillance of suspected artemisinin resistance parasites in Africa by providing baseline prevalence of k13-propeller mutations in western Kenya with samples collected from a longitudinal study. Clinical Trials Registration NCT00868465.

Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs

Graphical abstract ■■■. Research highlights ► A pyrimethamine+sulphadoxine-resistant rodent malaria genome was sequenced. ► Its genome sequence was compared with that of a sensitive progenitor. ► Four point mutations were identified: on chromosomes 2, 7, 13 and 14. ► The K392Q mutation in mdr2 is genetically linked to sulphadoxine resistance. ► mdr2 is a novel candidate gene underlying antifolate resistance in malaria.

The Mu Subunit of Plasmodium falciparum Clathrin-Associated Adaptor Protein 2 Modulates In Vitro Parasite Response to Artemisinin and Quinine

ABSTRACT The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

Plasmodium vivax in the Democratic Republic of East Timor: parasite prevalence and antifolate resistance-associated mutations.

In the Democratic Republic of East Timor, Plasmodium falciparum and Plasmodium vivax malaria coexist, but limited information is available about the latter species. Consequently, the prevalence of P. vivax and of its corresponding antifolate resistance-associated mutations in the pvdhfr and pvdhps genes was assessed here. Blood samples were collected from 650 individuals distributed among six districts, over two different periods, by either passive case detection (PCD) or active case detection (ACD). As expected, malaria was over-represented in the PCD sample (26% PCD vs 5% ACD), because the infection increases medical care seeking. Additionally, the relative frequency of P. vivax infections in symptomatic individuals (37%) was twice as high as the one in the asymptomatic sampling group (18%), suggesting that that this parasite is accounting for a significant proportion malaria-attributed morbidity. The frequency of specific sulfadoxine-pyrimethamine resistance-associated mutations genes was ascertained in P. vivax positive samples by PCR-RFLP. Although no mutants were detected in codons 383 and 553 of pvdhps, 48%, 76% and 82% of P. vivax-infected samples harbored the dhfr 33L, 58R and 117N mutations, respectively. Additionally, the frequency of parasites carrying both pvdhfr 58R and 117N mutant alleles accounted for a third of all genotypes analyzed, most likely due to inadvertent SP use in the past. In conclusion, evidence-based information is provided to promote optimized drug deployment and limit the evolution of resistance to antifolate resistance in P. vivax from East Timor.

MDR1-associated resistance to artesunate + mefloquine does not impair blood-stage parasite fitness in a rodent malaria model

If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin combination treatment (ACT) version of artesunate+mefloquine (ATN+MF) to gain insights about their ability to compete with ATN+MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN+MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN+MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.

Identification of genetic markers of resistance to Artemisinin Combination Therapy in the rodent model Plasmodium chabaudi

Background Effective treatment of malaria relies mostly on Artemisin Combination Therapy (ACT), which consists of the administration of an Artemisinin (ART) derivative in conjunction with a chemically unrelated anti-malarial, such as Mefloquine (MF). ACTs should reduce the chances of a parasite carrying mutations conferring resistance to both drugs [1]. However, parasites resistant to the different components of the combination have recently been reported. For instance, in Southeast Asia, the appearance of parasites showing increased in vivo tolerance to artesunate (ATN) [2,3] may undermine the future efficacy of the ATN+MF combination. We have previously selected stable resistance to ATN in the rodent malaria parasite Plasmodium chabaudi [4]. ATN-resistant parasites showed a mutation on pcubp1 gene, coding fora deubiquitinating enzyme [5]. No changes were found in thepcatp6 and and pcmdr1 genes [4]. In order to study the genetics of resistance to ACTs, the ATN-resistant P.chabaudi clone was repeatedly subinoculated into mice continuously treated with ATN + MF. Upon reaching a certain level of resistance, parasites were cloned by limiting dilution. The parasites’ genetic background was investigated by SOLEXA whole genome re-sequencing. The identified mutations were confirmed by dideoxy sequencing real-time polymerase chain reaction. Results Selection and cloning procedures originated five parasite clones, of which only one, denoted AS-ATNMF-1, was investigated. When compared to the ATN-resistant progenitor, AS-ATNMF-1 is resistant to treatment with the combination of both ATN+MF, as well as to each drug separately. AS-ATNMF-1 carries three distinctive mutations one of which is a duplication of the mdr1 gene. The remaining two mutations are SNPs in genes of unknown function and remain to be further investigated. No differences were found in the coding sequences of pcatp6 and pcubp1 when comparing AS-ATNMF-1 and its progenitor.