Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

Almeida

et al. 2018

Malar J

Self Cite

BackgroundThe resistance of Plasmodium vivax to chloroquine has become an obstacle to control strategies based on the use of anti-malarials. The current study investigated the association between P. vivax CQ-resistance in vivo with copy number variation and mutations in the promoter region in pvcrt-o and pvmdr1 genes.MethodsThe study included patients with P. vivax that received supervised treatment with chloroquine and primaquine. Recurrences were actively recorded during this period.ResultsAmong the 60 patients with P. vivax, 25 were CQ-resistant and 35 CQ-susceptible. A frequency of 7.1% of multi-copy pvcrt-o was observed in CQ-susceptible samples and 7.7% in CQ-resistant at D0 (P > 0.05) and 33.3% in CQ-resistant at DR (P < 0.05). For pvmdr1, 10.7% of the CQ-susceptible samples presented multiple copies compared to 11.1% in CQ-resistant at D0 and 0.0% in CQ-resistant at DR (P > 0.05). A deletion of 19 bp was found in 11/23 (47.6%) of the patients with CQ-susceptible P. vivax and 3/10 (23.1%) of the samples with in CQRPv at D0. At day DR, 55.5% of the samples with CQRPv had the 19 bp deletion. For the pvmdr-1 gene, was no variation in the analysed gene compared to the P. vivax reference Sal-1.ConclusionsThis was the first study with 42-day clinical follow-up to evaluate the variation of the number of copies and polymorphisms in the promoter region of the pvcrt-o and pvmdr1 genes in relation to treatment outcomes. Significantly higher frequency of multi-copy pvcrt-o was found in CQRPv samples at DR compared to CQ-susceptible, indicating parasite selection of this genotype after CQ treatment and its association with CQ-resistance in vivo.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2411-5) contains supplementary material, which is available to authorized users.

Section: Discussionmentioning

confidence: 99%

“…PCR products were amplified and identified on a 7500 FAST (Applied Biosystems). Sequences of primers used to determine copy number variation of pvcrt - o [ 22 ] and pvmdr - 1 [ 23 ] genes were described in Additional file 1 .…”

Section: Methodsmentioning

confidence: 99%

Chloroquine resistance is associated to multi-copy pvcrt-o gene in Plasmodium vivax malaria in the Brazilian Amazon

Almeida

et al. 2018

Malar J

Self Cite

“…Similarly, a 7–39% amplification rate was reported in Thailand [ 40 , 62 ]. There was no pvmdr1 amplification in Indonesia [ 36 , 67 ] and low prevalence in Brazil (0.9–4%) [ 67 , 68 ]. The lack or low rate of pvmdr1 amplification in some areas of Thailand and Cambodia with intense MQ pressure does not support previous evidence associating pvmdr1 amplification with MQ pressure [ 67 ].…”

Section: Discussionmentioning

confidence: 99%

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia

Chaorattanakawee

Lon

Chann

et al. 2017

Malar J

BackgroundWhile intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013–2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates.ResultsEx vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013–4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification.ConclusionThe findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-2034-2) contains supplementary material, which is available to authorized users.

“…In Ethiopia, the chloroquine resistance transporter gene ( Pvcrt ) of P. vivax on chromosome 14 had been shown with significant selection in a region upstream of the promotor, highlighting the ability of P. vivax to rapidly evolve in response to control measures [20]. Apart from mutations, high copy number observed in Pvcrt and multidrug resistant gene ( Pvmdr 1) has also been shown to be associated with increased antimalaria drug resistance [21,22].…”

Section: Introductionmentioning

confidence: 99%

Whole Genome Sequencing ofPlasmodium vivaxIsolates Reveals Frequent Sequence and Structural Polymorphisms in Erythrocyte Binding Genes

Ford

Kepple

Abagero

et al. 2020

Preprint

45 Plasmodium vivax malaria is much less common in Africa than the rest of the world 46 because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) 47 to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, 48 there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a 49 high number of them being in Duffy negative individuals, potentially indicating P. vivax 50 has evolved an alternative invasion mechanism that can overcome Duffy negativity.51 Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation 52 (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from 53 symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and 54 Duffy negative individuals are found. A total of 236,351 SNPs were detected, of which 55 21.9% was nonsynonymous and 78.1% was synonymous mutations. The largest 56 number of SNPs were detected on chromosomes 9 (33,478 SNPs; 14% of total) and 10 57 (28,133 SNPs; 11.9%). There were particularly high levels of polymorphism in 58 erythrocyte binding gene candidates including reticulocyte binding protein 2c (RBP2c), 59 merozoite surface protein 1 (MSP1), and merozoite surface protein 3 (MSP3.5, 60 MSP3.85 and MSP3.9). Thirteen genes related to immunogenicity and erythrocyte 61 binding function were detected with significant signals of positive selection. Variation in 62 gene copy number was also concentrated in genes involved in host-parasite 63 interactions, including the expansion of the Duffy binding protein gene (PvDBP) on 64 chromosome 6 and several PIR genes. Based on the phylogeny constructed from the 65 whole genome sequences, the expansion of these genes was an independent process 66 among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. 67 vivax infections among study sites and showed various levels of gene flow at a small 68 geographical scale. The genomic features of P. vivax provided baseline data for future 69 comparison with those in Duffy-negative individuals, and allowed us to develop a panel 70 of informative Single Nucleotide Polymorphic markers diagnostic at a micro-71 geographical scale. 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 90 Vivax malaria is the most geographically widespread human malaria, causing over 130 91 million clinical cases per year worldwide [1]. Plasmodium vivax can produce dormant 92 liver-stage hypnozoites within infected hosts, giving rise to relapse infections from 93 months to years. This unique feature of P. vivax contributes to an increase in 94 transmission potential and increases the challenge of elimination [2]. Understanding P.95 vivax genome variation will advance our knowledge of parasite biology and host-96 parasite interactions, as well as identify potential drug resistance mechanisms [3, 4].97 Such data will also help identify molecular targets for vaccine development [5][6][7], and 98 provide new means to track the transmission and spread of ...