scholarly journals Optimizing Bulk Segregant Analysis of Drug Resistance Using Plasmodium Falciparum Genetic Crosses Conducted in Humanized Mice

2021 ◽  
Author(s):  
Katelyn V. Brenneman ◽  
Xue Li ◽  
Sudhir Kumar ◽  
Elizabeth Delgado ◽  
Lisa A. Checkley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Bulk Segregant Analysis ◽  
Humanized Mice ◽  
Genetic Crosses

scholarly journals Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice

2021 ◽  
Author(s):  
Katelyn Vendrely Brenneman ◽  
Xue Li ◽  
Sudhir Kumar ◽  
Elizabeth Delgado ◽  
Lisa A. Checkley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Complex Traits ◽  
Bulk Segregant Analysis ◽  
Artemisinin Resistance ◽  
Humanized Mice ◽  
Drug Selection ◽  
Genetic Crosses ◽  
Parasite Plasmodium Falciparum ◽  
Trait Locus

Background: Classical genetic crosses in malaria parasites involve isolation, genotyping, and phenotyping of multiple progeny parasites, which is time consuming and laborious. Bulk segregant analysis (BSA) offers a powerful and efficient alternative to identify loci underlying complex traits in the human malaria parasite, Plasmodium falciparum. Methods: We have used BSA, which combines genetic crosses using humanized mice with pooled sequencing of progeny populations to measure changes in allele frequency following selection with antimalarial drugs. We used dihydroartemisinin (DHA) drug selection in two genetic crosses (Mal31xKH004 and NF54xNHP1337). We specifically investigated how synchronization, cryopreservation, and the drug selection regimen of progeny pools impacted the success of BSA experiments. Findings: We detected a strong and repeatable quantitative trait locus (QTL) at chr13 kelch13 locus in both crosses, but did not detect QTLs at ferredoxin (fd), the apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2). QTLs were detected using synchronized, but not unsynchronized pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools. Interpretation: Our results provide proof-of-principal of the utility of BSA for rapid, robust genetic mapping of drug resistance loci. Use of cryopreserved progeny pools expands the utility of BSA because we can conduct experiments using archived progeny pools from previous genetic crosses. BSA provides a powerful approach that complements traditional QTL methods for investigating the genetic architecture of resistance to antimalarials, and to reveal new or accessory loci contributing to artemisinin resistance.

scholarly journals Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

2019 ◽  
Author(s):  
Katrina A. Button-Simons ◽  
Sudhir Kumar ◽  
Nelly Carmago ◽  
Meseret T. Haile ◽  
Catherine Jett ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Human Liver ◽  
Statistical Power ◽  
Artemisinin Resistance ◽  
Southeast Asian ◽  
Geographic Region ◽  
Genetic Maps ◽  
Promising Alternative ◽  
Genetic Crosses

AbstractGenetic crosses are most powerful for linkage analysis when progeny numbers are high, when parental alleles segregate evenly and, for hermaphroditic organisms, when numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. Here we examine the statistical power of two different genetic crosses – (1) between a laboratory parasite (NF54) of African origin and a patient-derived Asian parasite, and (2) between two sympatric patient-derived Asian parasites. We generated >140 unique recombinant clones over a 12-month period from the four parental genotypes, doubling the number of unique recombinant progeny generated in the previous 30 years. Both crosses show bi-parental inheritance of plastid markers amongst recombinant progeny, in contrast to previous crosses (conducted using chimpanzee hosts) which carried single dominant plastid genotypes. Both crosses show distinctive segregation patterns. The allopatric African/Asian cross has minimal levels of inbreeding (2% of clonal progeny are inbred) and extreme skews in marker segregation, while in the sympatric Asian cross, inbred progeny predominate (66% of clonal progeny are inbred) and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny arrays (particularly the sympatric Asian cross) have excellent power to map large-effect mutations to a 31 kb interval and can capture complex, epistatic interactions that were far beyond the capacity of previous malaria crosses to detect. The extreme segregation distortion in the allopatric African/Asian cross erodes power to detect linkage in several genome regions, but the repeatable distortions observed offer promising alternative approaches to identifying genes underlying traits of interest. These crosses show surprising variation in marker segregation, nevertheless, the increased progeny numbers improve our ability to rapidly map biomedically important parasite traits.Author SummaryUnderstanding how genome mutations contribute to newly emerging drug resistance in parasites like Plasmodium falciparum is important to monitor the spread of drug resistance. This scenario has been playing out in Southeast Asia with the emergence and spread of artemisinin resistance. Here we show that new P. falciparum genetic crosses, using mice carrying human liver cells and infused with human red blood cells (the human liver-chimeric FRG NOD huHep/huRBC mouse), provide an important new tool for understanding complex interactions underlying drug resistance phenotypes. We report two new genetic maps with 84 and 60 unique recombinant progeny, which doubles the number of progeny available from 4 previous P. falciparum genetic crosses. Through extensive simulations we show that with 84 progeny we can find association for a gene that controls only 20% of the variation in a phenotype. We also show that a cross generated from Southeast Asian parasites collected from the same geographic region have unique characteristics not previously observed in P. falciparum genetic crosses. This Southeast Asian cross exhibits even segregation across the genome, unbiased inheritance of mitochondria and apicoplast and higher levels of inbreeding than previously observed.

scholarly journals Genome variation and meiotic recombination in Plasmodium falciparum: insights from deep sequencing of genetic crosses

2015 ◽  
Author(s):  
Alistair Miles ◽  
Zamin Iqbal ◽  
Paul Vauterin ◽  
Richard Pearson ◽  
Susana Campino ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Deep Sequencing ◽  
Meiotic Recombination ◽  
Web Application ◽  
Large Fraction ◽  
Error Rates ◽  
High Rate ◽  
Integrated Analysis ◽  
Genetic Crosses

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable, and thus difficult to analyse using short read technologies. Here we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, INDEL and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that INDELs are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and INDEL markers to analyse patterns of meiotic recombination, confirming a high rate of crossover events, and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of recombination that modify copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired. We describe a novel web application that allows these data to be explored in detail.

scholarly journals Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

2006 ◽  
Vol 50 (10) ◽  
pp. 3343-3349 ◽  
Author(s):  
Halima Kaddouri ◽  
Serge Nakache ◽  
Sandrine Houzé ◽  
France Mentré ◽  
Jacques Le Bras
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Lactate Dehydrogenase ◽  
Active Metabolite ◽  
Inhibitory Concentration ◽  
Drug Susceptibility ◽  
Maximum Effect ◽  
Malaria Parasites ◽  
Effect Model

ABSTRACT The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC50s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

Detection of Genetic markers associated with Plasmodium falciparum drug resistance among malaria patients in Kaduna State, Nigeria

2021 ◽  
Vol 42 (2) ◽  
pp. 206-213
Author(s):  
G.Y. Benjamin ◽  
H.I. Inabo ◽  
M.H.I. Doko ◽  
B.O. Olayinka
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Phylogenetic Tree ◽  
Genetic Markers ◽  
Cross Sectional Study ◽  
Sub Saharan Africa ◽  
Health Concern ◽  
Cross Sectional ◽  
Blood Samples ◽  
Test Kit

Malaria is a disease of public health concern in Nigeria and sub-Saharan Africa. It is caused by intracellular parasites of the genus Plasmodium. The aim of this study was to detect genetic markers associated with Plasmodium falciparum drug resistance among malaria patients in Kaduna State, Nigeria. The study was a cross-sectional study that lasted from May 2018 to October 2018. Three hundred blood samples were collected from consenting individuals attending selected hospitals, in the three senatorial districts of Kaduna State, Nigeria. Structured questionnaire were used to obtain relevant data from study participants. The blood samples were screened for malaria parasites using microscopy and rapid diagnostic test kit. Polymerase Chain Reaction was used for detection of the drug resistance genes. Pfcrt, pfmdr1, pfdhfr, pfdhps and pfatpase6 genes were detected at expected amplicon sizes from the malaria positive samples. The pfatpase6 PCR amplicons were sequenced and a phylogenetic tree was created to determine their relatedness. Result showed that Pfcrt (80%) had the highest prevalence, followed by pfdhfr (60%), pfmdr1 (36%) and pfdhps (8%). Pfatpase6 was also detected in 73.3% of the samples, and a phylogenetic tree showed relatedness between the pfatpase6  sequences in this study and those deposited in the GenBank. In conclusion, the study detected that Plasmodium falciparum genes were associated with drug resistance to commonly used antimalarials.

History and current status of Plasmodium falciparum antimalarial drug resistance in Madagascar

2010 ◽  
Vol 42 (1) ◽  
pp. 22-32 ◽  
Author(s):  
Valérie Andriantsoanirina ◽  
Didier Ménard ◽  
Luciano Tuseo ◽  
Rémy Durand
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Current Status ◽  
Antimalarial Drug Resistance

scholarly journals Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

2020 ◽  
Author(s):  
Nonlawat Boonyalai ◽  
Brian A Vesely ◽  
Chatchadaporn Thamnurak ◽  
Chantida Praditpol ◽  
Watcharintorn Fagnark ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Copy Number ◽  
Drug Susceptibility ◽  
Antagonistic Activity ◽  
Drug Combinations ◽  
Chloroquine Resistance ◽  
Phenotypic Characterization ◽  
Field Isolates

Abstract Background High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 ( pfpm2 ), exonuclease ( pfexo ) and chloroquine resistance transporter ( pfcrt ) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.Results The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovoquone-proguanil combinations revealed synergistic antimalarial activity.Conclusions Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

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