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

[This corrects the article DOI: 10.1371/journal.pone.

Geo-classification of drug-resistant travel-associated using and gene sequences (USA, 2018-2021).

Travel-related malaria is regularly encountered in the United States, and the U.S. Centers for Disease Control and Prevention (CDC) characterizes drug-resistance genotypes routinely for travel-related cases.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021.

Background: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021.

Methods: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021.

Simultaneous targeted amplicon deep sequencing and library preparation for a time and cost-effective universal parasite diagnostic sequencing approach.

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp.

Molecular Markers of Sulfadoxine-Pyrimethamine Resistance in Samples from Children with Uncomplicated Plasmodium falciparum at Three Sites in Angola in 2019.

Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase () and dihydropteroate synthase () genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools.

Portable and cost-effective genetic detection and characterization of Plasmodium falciparum hrp2 using the MinION sequencer.

The prevalence of Plasmodium falciparum hrp2 (pfhrp2)-deleted parasites threatens the efficacy of the most used and sensitive malaria rapid diagnostic tests and highlights the need for continued surveillance for this gene deletion. While PCR methods are adequate for determining pfhrp2 presence or absence, they offer a limited view of its genetic diversity. Here, we present a portable sequencing method using the MinION.

The first complete genome of the simian malaria parasite Plasmodium brasilianum.

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P.

Polymorphic Molecular Signatures in Variable Regions of the DBL3x Domain Are Associated with Virulence in Placental Malaria.

The protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood.

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.

Plasmodium falciparum kelch 13 Mutations, 9 Countries in Africa, 2014-2018.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018.

Therapeutic efficacy of artemether-lumefantrine and artesunate-amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Mali, 2015-2016.

Background: The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali.

Methods: Children between 6 and 59 months of age with uncomplicated Plasmodium falciparum infection and 2000-200,000 asexual parasites/μL of blood were enrolled, randomly assigned to either AL or ASAQ, and followed up for 42 days.

Targeted deep amplicon sequencing of antimalarial resistance markers in Plasmodium falciparum isolates from Cameroon.

Background: Recent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region.

Method: In this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum.

Results: No confirmed or associated artemisinin resistance markers were observed in Pfk13.

Association of Plasmodium falciparum kelch13 R561H genotypes with delayed parasite clearance in Rwanda: an open-label, single-arm, multicentre, therapeutic efficacy study.

Background: Partial artemisinin resistance is suspected if delayed parasite clearance (ie, persistence of parasitaemia on day 3 after treatment initiation) is observed. Validated markers of artemisinin partial resistance in southeast Asia, Plasmodium falciparum kelch13 (Pfkelch13) R561H and P574L, have been reported in Rwanda but no association with parasite clearance has been observed. We aimed to establish the efficacy of artemether-lumefantrine and genetic characterisation of Pfkelch13 alleles and their association with treatment outcomes.

Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing.

Background: Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging.

Continued Low Efficacy of Artemether-Lumefantrine in Angola in 2019.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers.

Malaria Risk and Prevention in Asian Migrants to Angola.

The number of Asian migrants working in sub-Saharan developing countries like Angola has been increasing. Their malaria risk, prevention, and care-seeking practices have not been characterized. A cross-sectional survey was conducted in 733 Chinese and Southeast Asian migrants in Angola.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C.

One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum.

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious.

Efficacy and safety of artesunate-amodiaquine and artemether-lumefantrine and prevalence of molecular markers associated with resistance, Guinea: an open-label two-arm randomised controlled trial.

Background: Anti-malarial resistance is a threat to recent gains in malaria control. This study aimed to assess the efficacy and safety of artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) in the management of uncomplicated malaria and to measure the prevalence of molecular markers of resistance of Plasmodium falciparum in sentinel sites in Maferinyah and Labé Health Districts in Guinea in 2016.

Methods: This was a two-arm randomised controlled trial of the efficacy of AL and ASAQ among children aged 6-59 months with uncomplicated Plasmodium falciparum malaria in two sites.

Development of a workflow for identification of nuclear genotyping markers for Cyclospora cayetanensis.

Cyclospora cayetanensis is an intestinal parasite responsible for the diarrheal illness, cyclosporiasis. Molecular genotyping, using targeted amplicon sequencing, provides a complementary tool for outbreak investigations, especially when epidemiological data are insufficient for linking cases and identifying clusters. The goal of this study was to identify candidate genotyping markers using a novel workflow for detection of segregating single nucleotide polymorphisms (SNPs) in C.

Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol.

Background: Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites.

Next Generation Sequencing and Bioinformatics Methodologies for Infectious Disease Research and Public Health: Approaches, Applications, and Considerations for Development of Laboratory Capacity.

Next generation sequencing (NGS) combined with bioinformatics has successfully been used in a vast array of analyses for infectious disease research of public health relevance. For instance, NGS and bioinformatics approaches have been used to identify outbreak origins, track transmissions, investigate epidemic dynamics, determine etiological agents of a disease, and discover novel human pathogens. However, implementation of high-quality NGS and bioinformatics in research and public health laboratories can be challenging.

Genotyping genetically heterogeneous infections to complement epidemiological case linkage.

Sexually reproducing pathogens such as Cyclospora cayetanensis often produce genetically heterogeneous infections where the number of unique sequence types detected at any given locus varies depending on which locus is sequenced. The genotypes assigned to these infections quickly become complex when additional loci are analysed. This genetic heterogeneity confounds the utility of traditional sequence-typing and phylogenetic approaches for aiding epidemiological trace-back, and requires new methods to address this complexity.