High Proportion of Genome-Wide Homology and Increased Pretreatment pvcrt Levels in Plasmodium vivax Late Recurrences: a Chloroquine Therapeutic Efficacy Study.

Chloroquine (CQ) is the first-line treatment for Plasmodium vivax malaria in most countries where malaria is endemic. Monitoring P. vivax CQ resistance (CQR) is critical but remains challenged by the difficulty to distinguish real treatment failure from reinfection or liver relapse. The therapeutic efficacy of CQ against uncomplicated P. vivax malaria was evaluated in Gia Lai Province, Vietnam. Sixty-seven patients were enrolled and followed for 42 days using microscopy and quantitative PCR. Adequate clinical and parasitological response (ACPR) was 100% (66/66) on day 28 but 75.4% (49/65) on day 42. Eighteen recurrences (27.7%) were detected, with a median time to recurrence of 42 days (interquartile range [IQR], 35 to 42) and blood CQ concentration of 98% identity by descent to paired day 0 samples. This study shows that CQ remained largely efficacious to treat P. vivax in Gia Lai; i.e., recurrences occurred late (>day 28) and in the presence of low blood CQ concentrations. However, the combination of both WGS and gene expression analysis (pvcrt) data with clinical data (PCT) allowed us to identify potential emergence of low-grade CQR, which should be closely monitored. (This study has been registered at ClinicalTrials.gov under identifier NCT02610686.)

Selective whole-genome sequencing of Plasmodium parasites directly from blood samples by nanopore adaptive sampling

Abstract: Whole-genome sequencing of Plasmodium is becoming an increasingly important tool for genomic surveillance of malaria. Due to the predominance of human DNA in a patient blood sample, time-consuming laboratory procedures are required to deplete human DNA or enrich Plasmodium DNA. Here, we investigated the potential of nanopore adaptive sampling to enrich Plasmodium falciparum reads while sequencing unenriched patient blood samples. To compare adaptive sampling versus regular sequencing on a MinION device, a dilution series consisting of 0%\u201384% P . falciparum DNA in human DNA was sequenced. Half of the flow cell channels were run in adaptive sampling mode, enriching for the P. falciparum reference genome, resulting in a three- to five-fold enrichment of P. falciparum bases in samples containing 0.1%\u20138.4% P. falciparum DNA. This finding was confirmed by sequencing three P. falciparum patient blood samples with common levels of parasitemia, that is, 0.1%, 0.2%, and 0.6% in adaptive mode. Their estimated enrichment was 5.8, 3.9, and 2.7, respectively, which was sufficient to cover at least 97% of the P. falciparum reference genome at a median depth of 5 (lowest parasitemia) or 355 (highest parasitemia). In all, 38 drug resistance loci were compared to Sanger sequencing results, showing high concordance, which suggests that the obtained sequencing data are of sufficient quality to address common clinical research questions for patients with parasitemias of 0.1% and higher. Overall, our results indicate that adaptive nanopore sequencing has the potential to replace more time-consuming Plasmodium enrichment protocols in the future

Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes

During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection

Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000–2016)

Abstract Emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACT) is a major challenge for Greater Mekong Subregion countries in their goal to eliminate malaria by 2030. Tools to efficiently monitor drug resistance beyond resource-demanding therapeutic efficacy studies are necessary. A custom multiplex amplicon sequencing assay based on Illumina technology was designed to target the marker of partial resistance to artemisinin (K13), five candidate modulators of artemisinin resistance, the marker of resistance to chloroquine (crt), and four neutral microsatellite loci. The assay was used to genotype 635 P. falciparum-positive blood samples collected across seven provinces of Vietnam and one of Cambodia between 2000 and 2016. Markers of resistance to artemisinin partner-drugs piperaquine (copy number of plasmepsin-2) and mefloquine (copy number of multidrug-resistance 1) were determined by qPCR. Parasite population structure was further assessed using a 101-SNP barcode. Validated mutations of artemisinin partial resistance in K13 were found in 48.1% of samples, first detection was in 2000, and by 2015 prevalence overcame > 50% in Central Highlands and Binh Phuoc province. K13-C580Y variant became predominant country-wide, quickly replacing an outbreak of K13-I543T in Central Highlands. Mutations in candidate artemisinin resistance modulator genes paralleled the trends of K13 mutants, whereas resistance to piperaquine and mefloquine remained low (≈ 10%) by 2015–2016. Genomic tools applied to malaria surveillance generate comprehensive information on dynamics of drug resistance and population structure and reflect drug efficacy profiles from in vivo studies

Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes

During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection

Uptake of intermittent preventive treatment and pregnancy outcomes: health facilities and community surveys in Chókwè district, southern Mozambique

Abstract Background Malaria in pregnancy leads to serious adverse effects on the mother and the child and accounts for 75,000–200,000 infant deaths every year. Currently, the World Health Organization recommends intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine–pyrimethamine (SP) at each scheduled antenatal care (ANC) visit. This study aimed to assess IPTp-SP coverage in mothers delivering in health facilities and at the community. In addition, factors associated with low IPTp-SP uptake and malaria adverse outcomes in pregnancy were investigated. Methods A community and a health facility-based surveys were conducted in mothers delivering in Chókwè district, southern Mozambique. Social-demographic data, malaria prevention practices and obstetric history were recorded through self-report and antenatal records. For women delivering at health facilities, a clinical examination of mother and child was performed, and malaria infection at delivery was determined by rapid diagnostic test, microscopy, quantitative PCR and placental histology. Results Of 1141 participants, 46.6, 30.2, 13.5 and 9.6% reported taking ≥ 3, two, one and none SP doses, respectively. Low IPTp uptake (< 3 doses) was associated with non-institutional deliveries (AOR = 2.9, P < 0.001), first ANC visit after week 28 (AOR = 5.4, P < 0.001), low awareness of IPTp-SP (AOR = 1.6, P < 0.002) and having no or only primary education (AOR = 1.3, P = 0.041). The overall prevalence of maternal malaria (peripheral and/or placental) was 16.8% and was higher among women from rural areas compared to those from urban areas (AOR = 1.9, P < 0.001). Younger age (< 20 years; AOR = 1.6, P = 0.042) and living in rural areas (AOR = 1.9, P < 0.001) were predictors of maternal malaria at delivery. Being primigravidae (AOR = 2.2, P = 0.023) and preterm delivery (AOR = 2.6, P < 0.001) predicted low birth weight while younger age was also associated with premature delivery (AOR = 1.4, P = 0.031). Conclusion The coverage for two and ≥ 3 doses of IPTp-SP is moderately higher than estimates from routine health facility records in Gaza province in 2015. However, this is still far below the national target of 80% for ≥ 3 doses. Ongoing campaigns aiming to increase the use of malaria prevention strategies during pregnancy should particularly target rural populations, increasing IPTp-SP knowledge, stimulate early visits to ANC, improve access to health services and the quality of the service provided