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

• Published in: PloS one Vol. 17; no. 1; p. e0262616
• Main Authors: Louha, Swarnali, Herman, Camelia, Gupta, Mansi, Patel, Dhruviben, Kelley, Julia, OH, Je-Hoon M., Guru, Janani, Lemoine, Jean F., Chang, Michelle A., Venkatachalam, Udhayakumar, Rogier, Eric, Talundzic, Eldin
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.01.2022; Public Library of Science (PLoS)
• Subjects: Animals; Antimalarials - pharmacology; Biology and Life Sciences; Blood; Causes of; Chloroquine; Density; Deoxyribonucleic acid; Diagnostic tests; Dihydrofolate reductase; Discordance; Disease control; Disease prevention; Disease transmission; DNA; DNA sequencing; Dried Blood Spot Testing - methods; Drug resistance; Drug Resistance - genetics; Drug therapy; Epidemiological Monitoring; Estimates; Evaluation; Gene frequency; Gene sequencing; Genes; Genetic aspects; Genetic testing; Genotyping; Haiti; Health aspects; Health facilities; Health surveillance; High-Throughput Nucleotide Sequencing - methods; High-Throughput Screening Assays - methods; Human subjects; Humans; Malaria; Malaria - epidemiology; Malaria, Falciparum - parasitology; MDR1 protein; Medicine and Health Sciences; Methods; Mutation; Nucleic Acid Amplification Techniques - methods; Nucleotide sequencing; P-Glycoprotein; Parasite resistance; Parasites; Parasites - genetics; Parasitic diseases; Patients; People and places; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - genetics; Plasmodium falciparum - pathogenicity; Polymerase chain reaction; Polymerase Chain Reaction - methods; Pools; Population genetics; Population studies; Population-based studies; Research and analysis methods; Sentinel health events; Sequence Analysis, DNA; Vector-borne diseases; Haiti; Atlanta Georgia; United States > US
• DOI: 10.1371/journal.pone.0262616
• ISSN: 1932-6203

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.