Global Spread of Mutant PfCRT and Its Pleiotropic Impact on Plasmodium falciparum Multidrug Resistance and Fitness

Our study defines the allelic distribution of pfcrt, an important mediator of multidrug resistance in Plasmodium falciparum, in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, pfcrt allele-specific ways. This study underscores the importance of actively monitoring pfcrt genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.

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

mBio ◽

10.1128/mbio.02093-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Carla Calçada ◽

Miguel Silva ◽

Vitória Baptista ◽

Vandana Thathy ◽

Rita Silva-Pedrosa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Southeast Asia ◽

Gene Copy ◽

Multidrug Resistance Protein ◽

Combination Therapies ◽

Transport Capacity ◽

Multidrug Resistance Protein 1 ◽

Content Type ◽

Resistance Protein

ABSTRACT 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.

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Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies potential targets of allele-specific immunity to clinical malaria

10.1101/2020.09.16.20196253 ◽

2020 ◽

Author(s):

Zalak Shah ◽

Myo T Naung ◽

Kara A Moser ◽

Matthew Adams ◽

Andrea G Buchwald ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Sequence Data ◽

Clinical Malaria ◽

Whole Genome Sequence ◽

Whole Genome ◽

Whole Genome Analysis ◽

Multiple Alleles ◽

Vaccine Candidates ◽

Genome Wide ◽

Allele Specific

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. This immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode likely targets of naturally acquired immunity and that should be further characterized for their potential as vaccine candidates.

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Role ofPfmdr1inIn VitroPlasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03494-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7032-7040 ◽

Cited By ~ 41

Author(s):

Nathalie Wurtz ◽

Bécaye Fall ◽

Aurélie Pascual ◽

Mansour Fall ◽

Eric Baret ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Antimalarial Drug ◽

Antimalarial Drugs ◽

Wild Type ◽

Antimalarial Drug Resistance ◽

Content Type ◽

Increased Susceptibility

ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms inPfmdr1(N86Y, Y184F, S1034C, N1042D, and D1246Y) andPfcrt(K76T) andin vitroresponses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174Plasmodium falciparumisolates from Dakar, Senegal. ThePfmdr186Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. ThePfmdr186Y mutation was significantly associated with increased susceptibility to MDAQ (P= 0.0023), LMF (P= 0.0001), DHA (P= 0.0387), and MQ (P= 0.00002). The N86Y mutation was not associated with CQ (P= 0.214) or QN (P= 0.287) responses. ThePfmdr1184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P= 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). ThePfmdr186Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P= 0.0136), LMF (P= 0.0019), and MQ (P= 0.0001). The additionalPfmdr186Y mutation increased significantly thein vitrosusceptibility to MDAQ (P< 0.0001), LMF (P< 0.0001), MQ (P< 0.0001), and QN (P= 0.0026) in wild-typePfcrtK76 parasites. The additionalPfmdr186Y mutation significantly increased thein vitrosusceptibility to CQ (P= 0.0179) inPfcrt76T CQ-resistant parasites.

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Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03337-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7390-7397 ◽

Cited By ~ 17

Author(s):

Maria Isabel Veiga ◽

Nuno S. Osório ◽

Pedro Eduardo Ferreira ◽

Oscar Franzén ◽

Sabina Dahlstrom ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Abc Transporter ◽

Parasite Clearance ◽

Antimalarial Drugs ◽

Multidrug Resistance Protein ◽

Content Type ◽

Resistance Protein

ABSTRACTPlasmodium falciparumhas 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 theApicomplexaparasites.P. falciparumgenome 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 studiedPfmrp2. The role ofPfmrp2polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of thePfmrp2genetic 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 thatPfmrp2harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identifiedPfmrp2polymorphisms with alteredin vitrosusceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggestedPfmrp2polymorphisms modulate the parasite'sin vitroresponse to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association within vivoparasite clearance. In conclusion, our study reveals that thePfmrp2gene is the most diverse ABC transporter known inP. falciparumwith a potential role in antimalarial drug resistance.

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Emerging erm(B)-Mediated Macrolide Resistance Associated with Novel Multidrug Resistance Genomic Islands in Campylobacter

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00153-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 7

Author(s):

Dejun Liu ◽

Weiwen Liu ◽

Ziquan Lv ◽

Junjie Xia ◽

Xing Li ◽

...

Keyword(s):

Multidrug Resistance ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Horizontal Transmission ◽

Treatment Options ◽

Macrolide Resistance ◽

Genomic Islands ◽

Campylobacter Coli ◽

Aminoglycoside Resistance ◽

Content Type

ABSTRACT The rapid dissemination of the macrolide resistance gene erm(B) will likely compromise the efficacy of macrolides as the treatment of choice for campylobacteriosis. More importantly, erm(B) is always associated with several multidrug resistance genomic islands (MDRGIs), which confer resistance to multiple other antimicrobials. Continuous monitoring of the emergence of erm(B) and analysis of its associated genetic environments are crucial for our understanding of macrolide resistance in Campylobacter. In this study, 290 Campylobacter isolates (216 Campylobacter coli isolates and 74 Campylobacter jejuni isolates) were obtained from 1,039 fecal samples collected in 2016 from pigs and chickens from three regions of China (344 samples from Guangdong, 335 samples from Shanghai, and 360 samples from Shandong). Overall, 74 isolates (72 C. coli isolates and 2 C. jejuni isolates) were PCR positive for erm(B). Combined with data from previous years, we observed a trend of increasing prevalence of erm(B) in C. coli. Pulsed-field gel electrophoresis analyses suggested that both clonal expansion and horizontal transmission were involved in the dissemination of erm(B) in C. coli, and three novel types of erm(B)-associated MDRGIs were identified among the isolates. Furthermore, 2 erm(B)-harboring C. jejuni isolates also contained an aminoglycoside resistance genomic island and a multidrug-resistance-enhancing efflux pump, encoded by RE-cmeABC. Antimicrobial susceptibility testing showed that most of the isolates were resistant to all clinically important antimicrobial agents used for the treatment of campylobacteriosis. These findings suggest that the increasing prevalence of erm(B)-associated MDRGIs might further limit treatment options for campylobacteriosis.

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Worldwide distribution and environmental origin of the Adelaide imipenemase (AIM-1), a potent carbapenemase in Pseudomonas aeruginosa

Microbial Genomics ◽

10.1099/mgen.0.000715 ◽

2021 ◽

Vol 7 (12) ◽

Author(s):

Anteneh Amsalu ◽

Sylvia A. Sapula ◽

Jonathan J. Whittall ◽

Bradley J. Hart ◽

Jan M. Bell ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Species ◽

Treatment Options ◽

Amino Acid Sequences ◽

Whole Genome Sequence ◽

Resistant Bacteria ◽

Metagenomic Sequencing ◽

Content Type ◽

Link Type ◽

Wastewater Samples

Carbapenems are potent broad-spectrum β-lactam antibiotics reserved for the treatment of serious infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa . The surge in P. aeruginosa resistant to carbapenems is an urgent threat, as very few treatment options remain. Resistance to carbapenems is predominantly due to the presence of carbapenemase enzymes. The assessment of 147 P . aeruginosa isolates revealed that 32 isolates were carbapenem non-wild-type. These isolates were screened for carbapenem resistance genes using PCR. One isolate from wastewater contained the Adelaide imipenemase gene (bla AIM-1) and was compared phenotypically with a highly carbapenem-resistant clinical isolate containing the bla AIM-1 gene. A further investigation of wastewater samples from various local healthcare and non-healthcare sources as well as river water, using probe-based qPCR, revealed the presence of the bla AIM-1 gene in all the samples analysed. The widespread occurrence of bla AIM-1 throughout Adelaide hinted at the possibility of more generally extensive spread of this gene than originally thought. A blast search revealed the presence of the bla AIM-1 gene in Asia, North America and Europe. To elucidate the identity of the organism(s) carrying the bla AIM-1 gene, shotgun metagenomic sequencing was conducted on three wastewater samples from different locations. Comparison of these nucleotide sequences with a whole-genome sequence of a P. aeruginosa isolate revealed that, unlike the genetic environment and arrangement in P. aeruginosa , the bla AIM-1 gene was not carried as part of any mobile genetic elements. A phylogenetic tree constructed with the deduced amino acid sequences of AIM-1 suggested that the potential origin of the bla AIM-1 gene in P. aeruginosa might be the non-pathogenic environmental organism, Pseudoxanthomonas mexicana .

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Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00835-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4719-4726 ◽

Cited By ~ 174

Author(s):

Rithea Leang ◽

Walter R. J. Taylor ◽

Denis Mey Bouth ◽

Lijiang Song ◽

Joel Tarning ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Falciparum Malaria ◽

Significant Risk ◽

Artemisinin Resistance ◽

Plasmodium Falciparum Malaria ◽

Significant Risk Factor ◽

Open Label ◽

Content Type

ABSTRACTWestern Cambodia is recognized as the epicenter ofPlasmodium falciparummultidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested forin vitrosusceptibilities to piperaquine and mefloquine, polymorphisms in theK13gene, and the copy number of thePfmdr-1gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P<10−3). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC50) of PP were independent of treatment outcomes, in contrast to median mefloquine IC50, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM;P= 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying theK13mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308;P= 0.04). Our data show evidence ofP. falciparumresistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.)

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Allele-specific, nested, one tube PCR: application to Pfmdr1 polymorphisms in Plasmodium falciparum

Parasitology ◽

10.1017/s0031182099004382 ◽

1999 ◽

Vol 119 (1) ◽

pp. 1-6 ◽

Cited By ~ 9

Author(s):

I. S. ADAGU ◽

D. C. WARHURST

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Single Nucleotide Polymorphisms ◽

Resistance Gene ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Multidrug Resistance Gene ◽

Single Nucleotide ◽

Reliable Detection ◽

Allele Specific

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P. falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

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Immune Characterization of Plasmodium falciparum Parasites with a Shared Genetic Signature in a Region of Decreasing Transmission

Infection and Immunity ◽

10.1128/iai.01979-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 276-285 ◽

Cited By ~ 8

Author(s):

Amy K. Bei ◽

Ababacar Diouf ◽

Kazutoyo Miura ◽

Daniel B. Larremore ◽

Ulf Ribacke ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Clonal Diversity ◽

Clinical Isolates ◽

Immune Recognition ◽

Content Type ◽

Reverse Transcription Pcr ◽

Genetic Signatures ◽

Parasite Populations ◽

Specific Parasite

As the intensity of malaria transmission has declined,Plasmodium falciparumparasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. SinceP. falciparumEMP-1 (PfEMP-1) is a major variant surface antigen, we usedvarUps quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize thevargenes expressed by CGS parasites after short-termin vitroculture. CGS parasites show upregulation of UpsAvargenes and 2-cysteine-containing PfEMP-1 molecules and express the same dominantvartranscript. Our work indicates that the CGS parasites in this cluster express similarvargenes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population.

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Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies possible targets of allele-specific immunity to clinical malaria

PLoS Genetics ◽

10.1371/journal.pgen.1009576 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009576

Author(s):

Zalak Shah ◽

Myo T. Naung ◽

Kara A. Moser ◽

Matthew Adams ◽

Andrea G. Buchwald ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Sequence Data ◽

Clinical Malaria ◽

Whole Genome Sequence ◽

Whole Genome ◽

Whole Genome Analysis ◽

Multiple Alleles ◽

Vaccine Candidates ◽

Genome Wide ◽

Allele Specific

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. Immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode possible targets of naturally acquired immunity that should be validated immunologically and further characterized for their potential as vaccine candidates.

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