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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
- Source :
- PLoS ONE, Vol 17, Iss 1 (2022), PLoS ONE, PLoS ONE, Vol 17, Iss 1, p e0262616 (2022)
- Publication Year :
- 2022
- Publisher :
- Public Library of Science (PLoS), 2022.
-
Abstract
- 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.
- Subjects :
- Plasmodium
Quantitative Parasitology
Molecular biology
Single Nucleotide Polymorphisms
Drug Resistance
Gene Sequencing
Parasitemia
Polymerase Chain Reaction
Geographical locations
Medical Conditions
Sequencing techniques
Medicine and Health Sciences
DNA sequencing
Malaria, Falciparum
Multidisciplinary
Drugs
High-Throughput Nucleotide Sequencing
Gene Pool
Epidemiological Monitoring
Medicine
Nucleic Acid Amplification Techniques
Research Article
Science
Plasmodium falciparum
Antimalarials
Parasite Groups
Genetics
Parasitic Diseases
Animals
Humans
Parasites
Caribbean
Pharmacology
Evolutionary Biology
Population Biology
Biology and Life Sciences
Sequence Analysis, DNA
Tropical Diseases
Haiti
Malaria
High-Throughput Screening Assays
Research and analysis methods
Molecular biology techniques
North America
Parasitology
Dried Blood Spot Testing
People and places
Apicomplexa
Population Genetics
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 17
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....99300695e586fca848ea3b627e541403