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

ABSTRACT: 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 <100 ng/ml. Primary infections leading to recurrence occurred in younger individuals (median age for ACPR = 25 years [IQR, 20 to 28]; recurrences = 18 [16 to 21]; P = 0.002) had a longer parasite clearance time (PCT for ACPR = 47.5 h [IQR, 36.2 to 59.8 h]; recurrences = 54.2 [48.4 to 62.0]; P = 0.035) and higher pvcrt gene expression (median relative expression ratio for ACPR = 0.09 [IQR, 0.05 to 0.22]; recurrences = 0.20 [0.15 to 0.56]; P = 0.002), but showed no differences in ex vivo CQ sensitivity. Parasite genotyping by microsatellites, single nucleotide polymorphism (SNP) barcoding, and whole-genome sequencing (WGS) identified a majority of homologous recurrences, with 80% (8/10) showing >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.).

SUBMITTER: Rovira-Vallbona E

PROVIDER: S-EPMC8284456 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe efficacy of chloroquine treatment for vivax malaria has been rarely evaluated due to a lack of an appropriate testing method. The objective of this study was to conduct molecular monitoring of chloroquine resistance in Plasmodium vivax strains from vivax malaria patients in Yunnan Province, focusing on the analysis of polymorphism in the P. vivax chloroquine resistance transporter protein orthologous gene (pvcrt-o).MethodsIn accordance with the principles of a cohort study, blood samples were collected from malaria cases diagnosed with a P. vivax mono-infection in Yunnan Province from 2020 to 2022. Segmental PCR was used to amplify the whole pvcrt-o gene in the blood samples and their products were subsequently sequenced. The sequencing data were arranged to obtain the full coding DNA sequence (CDS) as well as the gene's promoter region sequences. The CDSs were aligned with the reference sequence (XM_001613407.1) of the P. vivax SalI isolate to identify the mutant loci.ResultsFrom a total of 375 blood samples taken from vivax malaria cases, 272 both whole gene CDSs (1272-1275 bp) and promoter DNA sequences (707 bp) of pvcrt-o gene were obtained. Among the whole CDSs, there were 7 single nucleotide polymorphic sites in which c.7 A>G was the minor allele frequency (MAF) site with 4.4% (12/272) detection rate. The mutation detection rate showed a significant decrease from 9.8% (10/102) in 2020 to 1.1% (1/92) in 2021 and 1.3% (1/78) in 2022, indicating statistical significance (χ2 = 11.256, P < 0.05). Among the identified 12 haplotypes, the majority of which were wild type (75.7%; 206/272). These four mutant haplotypes (Hap_3, Hap_5, Hap_9, and Hap_10) were classified as "K10 insertion type" and accounted for 12.1% (33/272). The detection rate of Hap_3 increased from 1.0% (1/102) in 2020 to 13.0% (12/92) in 2021 and 14.1% (11/78) in 2022, indicating statistical significance. A total of 23.8% (65/272) of the samples exhibited 14 bp (bp) deletions in the promoter region, occurring most frequently in the wild type haplotype (Hap_1) samples at a rate of 28.6% (59/206).ConclusionsIn recent years in Yunnan Province, a notable proportion of vivax malaria patients are infected by P. vivax strains with a "K10 insertion" and partial sequence deletions in the promoter region of the pvcrt-o gene, necessitating vigilance.

Project description:BACKGROUND: There are increasing reports of severe clinical cases exclusively associated with Plasmodium vivax infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance. PATIENTS: Two different patients presented at the Hospital Clinic in Barcelona with P. vivax malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000. METHODS: To exclude the possibility that the patient's severe symptoms were due to Plasmodium falciparum, a nested PCR was performed. A magnetic method was used to purify P. vivax free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in P. vivax, namely the P. vivax chloroquine resistance transporter, pvcrt-o, and the P. vivax multidrug resistance transporter, pvmdr 1. RESULTS: Results demonstrated that the severe clinical symptoms were exclusively due to P. vivax. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in pvmdr1 levels and up to 21.9-fold increase in pvcrt-o levels compared to expression levels of parasites from the other patients with mild symptoms. CONCLUSION: This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of pvmdr1 and particularly pvcrt-o expression levels as molecular markers of severe disease in P. vivax.

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

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