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

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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Molecular Surveillance of Plasmodium falciparum Drug Resistance Markers in Clinical Samples from Botswana

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.18-0440 ◽

2018 ◽

Vol 99 (6) ◽

pp. 1499-1503 ◽

Cited By ~ 2

Author(s):

Leabaneng Tawe ◽

Michela Menegon ◽

Pleasure Ramatlho ◽

Charles W. Muthoga ◽

Naledi Mutukwa ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Clinical Samples ◽

Molecular Surveillance ◽

Resistance Markers

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Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00552-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 1

Author(s):

Ruimin Zhou ◽

Chengyun Yang ◽

Suhua Li ◽

Yuling Zhao ◽

Ying Liu ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Henan Province ◽

Nucleotide Polymorphisms ◽

Combination Therapies ◽

Antimalarial Drug Resistance ◽

Single Nucleotide ◽

Molecular Surveillance ◽

Content Type

ABSTRACT Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

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Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

BMC Research Notes ◽

10.1186/s13104-020-05334-5 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Monday Tola ◽

Olumide Ajibola ◽

Emmanuel Taiwo Idowu ◽

Olusesan Omidiji ◽

Samson Taiwo Awolola ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug Resistance ◽

Allelic Discrimination ◽

Molecular Surveillance ◽

Malaria Burden ◽

Two Samples ◽

Southwest Nigeria ◽

Resistance Markers ◽

Drug Interventions

Abstract Objective Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1–61 years old from South-west Nigeria. Results Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.

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Molecular surveillance of antimalarial drug resistance related genes in Plasmodium falciparum isolates from Eritrea

Acta Tropica ◽

10.1016/j.actatropica.2016.02.007 ◽

2016 ◽

Vol 157 ◽

pp. 158-161 ◽

Cited By ~ 6

Author(s):

Michela Menegon ◽

Abduselam M. Nurahmed ◽

Albadawi A. Talha ◽

Bakri Y.M. Nour ◽

Carlo Severini

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Antimalarial Drug Resistance ◽

Molecular Surveillance

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Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02116-16 ◽

2017 ◽

Vol 61 (3) ◽

Cited By ~ 20

Author(s):

Eldin Talundzic ◽

Yaye D. Ndiaye ◽

Awa B. Deme ◽

Christian Olsen ◽

Dhruviben S. Patel ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Deep Sequencing ◽

Artemisinin Resistance ◽

Potential Drug ◽

Drug Pressure ◽

Low Frequencies ◽

Content Type ◽

Public Health Activity ◽

Codon Positions

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

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.19-0142 ◽

2019 ◽

Vol 101 (5) ◽

pp. 1139-1143

Author(s):

Nicholas F. Brazeau ◽

Ashenafi Assefa ◽

Hussein Mohammed ◽

Heven Seme ◽

Abeba G. Tsadik ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Deep Sequencing

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Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria.

10.21203/rs.3.rs-35555/v3 ◽

2020 ◽

Author(s):

Monday Tola ◽

Olumide Ajibola ◽

Taiwo Emmanuel Idowu ◽

Olusesan Omidiji ◽

Samson Taiwo Awolola ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug Resistance ◽

Allelic Discrimination ◽

Molecular Surveillance ◽

Malaria Burden ◽

Two Samples ◽

Southwest Nigeria ◽

Resistance Markers ◽

Drug Interventions

Abstract ObjectiveNigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. ResultsTwo Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.

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Molecular Surveillance of Antimalarial Resistance Pfcrt, Pfmdr1 and Pfk13 Polymorphisms in African Plasmodium Falciparum Imported Parasites to Wuhan, China

10.21203/rs.3.rs-87992/v1 ◽

2020 ◽

Author(s):

Cheng Weijia ◽

Xiaonan Song ◽

Huabing Tan ◽

Kai Wu ◽

Jian Li

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Mixed Type ◽

Antimalarial Drugs ◽

Wild Type ◽

Mutant Type ◽

Molecular Surveillance ◽

Severe Problem ◽

Antimalarial Resistance ◽

Effective Drugs

Abstract Background: The development of drug resistance in Plasmodium falciparum becomes a severe problem for malaria control globally. Before finding a practical solution, monitoring the susceptibility of P. falciparum resistance-related genes is crucial. It will offer valuable information on the drug resistance in malaria-endemic areas and guides the rational clinical use of antimalarial drugs.Methods：Filter paper blood was taken from patients with positive P. falciparum during 2017-2019 in Wuhan, China. The target fragments from pfcrt, pfmdr1, and k13 propeller (pfk13) genes of P. falciparum were amplified and sequenced. Subsequently, the polymorphisms of pfcrt, pfmdr1, and pfk13 and the haplotypes of Pfcrt and Pfmdr1 were analyzed.Results: Totally, 106 samples were collected. Subsequently, 98.11% (104/106), 100% (106/106), and 86.79% (92/106) of these samples were successfully amplified and sequenced for the pfcrt, pfmdr1, and pfk13 genes, respectively. The prevalence of Pfcrt K76T, Pfmdr1 N86Y, and Pfmdr1 Y184F mutation were 9.62%, 4.72%, and 47.17%, respectively. At codons 72-76 of pfcrt gene locus were showed three haplotypes, including CVMNK (wild-type), CVIET (mutation type), CV M/I N/E K/T (mixed type), with 87.50%, 9.62%, and 2.88% prevalence, respectively. For the pfmdr1 gene, including NY (wild type), NF and YF (mutant type), N Y/F, Y Y/F, and N/Y Y/F (mixed type), accounted for 34.91%, 43.40%, 3.77%, 15.09%, 0.94%, and 1.89%, respectively. A total of eleven Pfcrt/Pfmdr1 combined haplotypes, including six types of combined haplotypes, and five combined haplotypes with mixed-type, For pfk13, no mutation was detected. Conclusions: The wild-type SNPs and haplotypes for the pfcrt, and pfmdr1 genes become predominant in the current study. It indicates these isolates entirely or partly recovery their susceptibility to antimalarial drugs, including chloroquine, amodiaquine, and mefloquine. Moreover, it demonstrates these drugs can continue to be effective drugs for P. falciparum malaria cases treatment in Africa. Although no mutation is detected in pfk13, continuous molecular surveillance is still urgently necessary.

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Role of Known Molecular Markers of Resistance in the Antimalarial Potency of Piperaquine and Dihydroartemisinin In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01656-08 ◽

2009 ◽

Vol 53 (4) ◽

pp. 1362-1366 ◽

Cited By ~ 28

Author(s):

Sant Muangnoicharoen ◽

David J. Johnson ◽

Sornchai Looareesuwan ◽

Srivicha Krudsood ◽

Stephen A. Ward

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Artemisinin Combination Therapy ◽

Parasite Line ◽

South American ◽

Resistance Markers ◽

Positive Correlations ◽

The Individual

ABSTRACT Using a range of laboratory-adapted and genetically modified Plasmodium falciparum parasite isolates, we investigated the interaction between dihydroartemisinin and piperaquine (PIP), the individual components of an artemisinin combination therapy currently under development, in addition to the role of known drug resistance genes in parasite susceptibility in vitro. All but one parasite line investigated displayed an interaction of dihydroartemisinin and PIP that was antagonistic, although the degree of antagonism was isolate dependent. In terms of resistance markers, the pfcrt haplotypes CVIET and SVMNT were positively associated with reduced sensitivity to PIP, with parasites carrying the South American CQR (SVMNT) allele being generally less sensitive than CVIET parasites. Parasites carrying the CQS (CVMNK) allele displayed a further increase in PIP sensitivity compared with CVIET and SVMNT parasites. Our data indicate that PIP sensitivity was not affected by pfmdr1 sequence status, despite positive correlations between the structurally related compound amodiaquine and pfmdr1 mutations in other studies. In contrast, neither the pfcrt nor pfmdr1 sequence status had any significant impact on susceptibility to dihydroartemisinin.

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