Whole genome sequencing identifies novel mutations in malaria parasites resistant to artesunate (ATN) and to ATN + mefloquine combination.

Introduction: The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease. Methods: Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine. Results and discussion: Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.

Cerebral Malaria Model Applying Human Brain Organoids.

Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood-brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.

Revolutionizing Disease Modeling: The Emergence of Organoids in Cellular Systems.

Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells (iPSCs) technology, solved the complications associated with human embryonic stem cells (hESC) usage. Moreover, iPSCs made significant contributions for human medicine, such as in diagnosis, therapeutic and regenerative medicine. The two-dimensional (2D) models allowed for monolayer cellular culture in vitro; however, they were surpassed by the three-dimensional (3D) cell culture system. The 3D cell culture provides higher cell-cell contact and a multi-layered cell culture, which more closely respects cellular morphology and polarity. It is more tightly able to resemble conditions in vivo and a closer approach to the architecture of human tissues, such as human organoids. Organoids are 3D cellular structures that mimic the architecture and function of native tissues. They are generated in vitro from stem cells or differentiated cells, such as epithelial or neural cells, and are used to study organ development, disease modeling, and drug discovery. Organoids have become a powerful tool for understanding the cellular and molecular mechanisms underlying human physiology, providing new insights into the pathogenesis of cancer, metabolic diseases, and brain disorders. Although organoid technology is up-and-coming, it also has some limitations that require improvements.

Aesthetic Contouring of the Chest wall with Rib Resection.

BACKGROUND: In past decades, several invasive and noninvasive aesthetic procedures have been sought as a way to improve body contouring, and one may resort to uncertified and potentially dangerous procedures to achieve that goal. An example of this practice would be the resection of ribs to reduce the waist for aesthetic reasons. The objective was to check scientific evidence on the effectiveness and safety of removal of floating ribs for aesthetic purposes. METHODS: A systematic review of the literature was carried out in EMBASE/Elsevier, Cochrane, Scopus, Medline, PubMed, BVS, SciELO, OneFile, and Lilacs, throughout the period until June 2020, using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. RESULTS: Fifty-six articles were found in all databases. After applying the inclusion criteria, by reading the title and abstract of the studies found, only two articles were definitively included. One addressed the possibility of removing the 7th and 8th ribs for improving body contouring in patients with an exaggerated anterior projection of the chest wall, without showing cases or surgical techniques. The other demonstrated procedures of body contouring in transgender by the removal of the 11th and 12th ribs in five of the 22 patients studied, just providing a brief description of the surgical technique used, without details. CONCLUSIONS: Despite the relevance of the theme and its popularity, there is not enough scientific evidence to support the practice, effectiveness and safety of the resection of ribs for aesthetic purposes. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Expansion of a Specific Plasmodium falciparum PfMDR1 Haplotype in Southeast Asia with Increased Substrate Transport.

Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia.IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

Plasmodium falciparum Plasmepsin 2 Duplications, West Africa.

Dihydroartemisinin/piperaquine (DHA/PPQ) is increasingly deployed as antimalaria drug in Africa. We report the detection in Mali of Plasmodium falciparum infections carrying plasmepsin 2 duplications (associated with piperaquine resistance) in 7/65 recurrent infections within 2 months after DHA/PPQ treatment. These findings raise concerns about the long-term efficacy of DHA/PPQ treatment in Africa.

Complex polymorphisms in the Plasmodium falciparum multidrug resistance protein 2 gene and its contribution to antimalarial response.

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.

A new molecular surveillance system for leishmaniasis.

Presently, global efforts are being made to control and eradicate the deadliest tropical diseases through the improvement of adequate interventions. A critical point for programs to succeed is the prompt and accurate diagnosis in endemic regions. Rapid diagnostic tests (RDTs) are being massively deployed and used to improve diagnosis in tropical countries. In the present report, we evaluated the hypothesis of, after use for diagnosis, the reuse of the Leishmania RDT kit as a DNA source, which can be used downstream as a molecular surveillance and/or quality control tool. As a proof of principle, a polymerase chain reaction-based method was used to detect Leishmania spp. minicircle kinetoplast DNA from leishmaniasis RDT kits. Our results show that Leishmania spp. DNA can be extracted from used RDTs and may constitute an important, reliable, and affordable tool to assist in future leishmaniasis molecular surveillance methods.

Single nucleotide polymorphisms in Plasmodium falciparum V type H(+) pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms.

BACKGROUND: Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H(+)). V type H(+) pyrophosphatase (PfVP2) is thought to pump H(+) into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms. METHODS: Blood samples from 384 patients collected (1981-2009) in Honduras (n=35), Colombia (n=50), Liberia (n=50), Guinea Bissau (n=50), Tanzania (n=50), Iran (n=50), Thailand (n=49) and Vanuatu (n=50) were analysed. The pfcrt 72-76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods. RESULTS: Pfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P=0.01) and Liberia+Guinea-Bissau (P=0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P=0.007). CONCLUSIONS: The VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.

Artemisinin resistance in Plasmodium falciparum: what is it really?

Until very recently, artemisinin and its derivatives were the only commercially available antimalarial drugs for which there was no reported parasite resistance. Artemisinin combination therapies (ACTs) are currently relied upon for effective malaria treatment in most regions of the world in which the disease is endemic, and their continuing efficacy is crucial if control and elimination programmes are to succeed. The loss of effectiveness of artemisinin and its derivatives to drug resistance would constitute a major disaster in the fight against malaria. To properly assess the danger posed by artemisinin resistance, and therefore enable appropriate and proportionate responses, definitions of 'artemisinin resistance' and 'ACT resistance', at both the clinical and parasitological levels, are needed.

Assessing the cost-benefit effect of a Plasmodium falciparum drug resistance mutation on parasite growth in vitro.

Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.

PfMDR1: mechanisms of transport modulation by functional polymorphisms.

ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance.

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

Antimalarial exposure delays Plasmodium falciparum intra-erythrocytic cycle and drives drug transporter genes expression.

BACKGROUND: Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in eukaryotic systems, e.g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS: We therefore investigated the effect of mefloquine exposure on the cell cycle of three P. falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold. CONCLUSIONS/SIGNIFICANCE: Both cell cycle delay and induced gene expression represent potentially important mechanisms for parasites to escape the effect of the antimalarial drug.

Polymorphism of antimalaria drug metabolizing, nuclear receptor, and drug transport genes among malaria patients in Zanzibar, East Africa.

Artemisinin-based combination therapy is a main strategy for malaria control in Africa. Zanzibar introduced this new treatment policy in 2003. The authors have studied the prevalence of a number of functional single nucleotide polymorphisms (SNPs) in genes associated with the elimination of the artemisinin-based combination therapy compounds in use in Zanzibar to investigate the frequencies of subgroups potentially at higher drug exposure and therefore possible higher risk of toxicity. One hundred three unrelated children with uncomplicated malaria from the Unguja and Pemba islands of Zanzibar were enrolled. With use of polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time PCR-based allele discrimination methods, the CYP2B6 (G15631T), CYP3A4 (A-392G), CYP3A5 (A6986G, G14690A, 27131-132 insT, C3699T) SNPs and MDR1 SNPs C3435T, G2677T/A, and T-129C were analyzed. PCR product sequencing was applied to regulatory regions of MDR1, the CYP3A4 proximal promoter, and to exons 2 and 5 of PXR, a gene coding for a nuclear factor activated by artemisinin antimalarials and associated with the transcription induction of most of the studied genes. Homozygous subjects for alleles coding for low activity proteins were found at the following frequencies: 1) MDR1: 2.9%; 2) CYP2B6: 9.7%; 3) CYP3A5: 14.1%; and 4) CYP3A4: 49.5%. No functionally relevant allele was found in the analyzed regions of PXR. A new MDR1 SNP was found (T-158C), located in a putative antigen recognition element. Ten (10.1%) subjects were predicted to be low metabolizers simultaneously for CYP3A4 and CYP3A5. This fraction of the population is suggested to be under higher exposure to certain antimalarials, including lumefantrine and quinine.

Pharmacogenetic tools for malaria and TB in the Developing World.

Some of the largest therapeutic drug exposures in the planet involve drugs employed against malaria and TB, two main global infectious diseases. Amodiaquine for malaria and isoniazid for TB are two pivotal drugs in the management of these diseases. Both drugs have been associated with severe adverse events. Amodiaquine and isoniazid are metabolized polymorphically by CYP2C8 and N-acetyltransferase 2, respectively. The polymorphic genes coding for these enzymes presently represent the best candidates for the application of personal pharmacogenetics for these diseases. We review the main reasons for this view, while asking the pivotal question of whether it is presently possible for pharmacogenetic-based personalized medicine to be applied in the malaria and TB settings of the Developing World.

Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum.

Plasmodium falciparum drug resistance is a major factor for the death toll of malaria. Resistance has been associated with specific single nucleotide polymorphisms (SNPs) in the parasite genes pfmdr1 (N86Y) and pfcrt (K76T) associated with quionoline antimalarial resistance, and pfdhfr (N51I, C59R, S108N) correlated with resistance of the antifolate combination sulfadoxine-pyrimethamine. These SNPs constitute the basis for the surveillance of drug resistance through high sensitive molecular methods in malaria endemic countries. In this work, we developed a multiplex PCR-RFLP protocols for the diagnosis of these molecular markers, leading to significant decreases in reagent costs, time, number of manipulations and hence human resources.