Pooled Deep Sequencing of Drug Resistance Loci from Plasmodium falciparum Parasites across Ethiopia

ABSTRACT The emergence of Plasmodium falciparum resistance to artemisinin in Southeast Asia threatens malaria control and elimination activities worldwide. Multiple polymorphisms in the P. falciparum kelch gene found in chromosome 13 (Pfk13) have been associated with artemisinin resistance. Surveillance of potential drug resistance loci within a population that may emerge under increasing drug pressure is an important public health activity. In this context, P. falciparum infections from an observational surveillance study in Senegal were genotyped using targeted amplicon deep sequencing (TADS) for Pfk13 polymorphisms. The results were compared to previously reported Pfk13 polymorphisms from around the world. A total of 22 Pfk13 propeller domain polymorphisms were identified in this study, of which 12 have previously not been reported. Interestingly, of the 10 polymorphisms identified in the present study that were also previously reported, all had a different amino acid substitution at these codon positions. Most of the polymorphisms were present at low frequencies and were confined to single isolates, suggesting they are likely transient polymorphisms that are part of naturally evolving parasite populations. The results of this study underscore the need to identify potential drug resistance loci existing within a population, which may emerge under increasing drug pressure.

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Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

PLoS ONE ◽

10.1371/journal.pone.0262616 ◽

2022 ◽

Vol 17 (1) ◽

pp. e0262616

Author(s):

Swarnali Louha ◽

Camelia Herman ◽

Mansi Gupta ◽

Dhruviben Patel ◽

Julia Kelley ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Deep Sequencing ◽

Parasite Density ◽

Population Level ◽

Dried Blood Spots ◽

Time Saving ◽

Molecular Surveillance ◽

The Individual ◽

Pooled Samples

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

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Pooled Deep Sequencing of Plasmodium falciparum Isolates: An Efficient and Scalable Tool to Quantify Prevailing Malaria Drug-Resistance Genotypes

The Journal of Infectious Diseases ◽

10.1093/infdis/jit392 ◽

2013 ◽

Vol 208 (12) ◽

pp. 1998-2006 ◽

Cited By ~ 36

Author(s):

Steve M. Taylor ◽

Christian M. Parobek ◽

Nash Aragam ◽

Billy E. Ngasala ◽

Andreas Mårtensson ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Deep Sequencing

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

10.1101/024182 ◽

2015 ◽

Cited By ~ 5

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.

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Method for Evaluation of Drug Resistance for Plasmodium Falciparum. Phase 2

10.21236/adb158380 ◽

1991 ◽

Author(s):

Michael T. Makler

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Phase 2

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00367-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3343-3349 ◽

Cited By ~ 61

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.

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

Nigerian Journal of Parasitology ◽

10.4314/njpar.v42i2.3 ◽

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.

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