Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry

Wirjanata, Grennady; Handayuni, Irene; Prayoga, Pak; Apriyanti, Dwi; Chalfein, Ferryanto; Sebayang, Boni F.; Kho, Steven; Noviyanti, Rintis; Kenangalem, Enny; Campo, Brice; Poespoprodjo, Jeanne Rini; Price, Ric N.; Marfurt, Jutta

doi:10.1186/s12936-015-0940-8

Cited by 16 publications

(15 citation statements)

References 38 publications

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“…vivax sensitivity to pCQ on the same isolates used to examine the asexual maturation inhibition with IVM. These results showed pCQ IC 50 values ranging from 1.96 ng/mL to 7.53ng/mL, similar to other reports [ 66 – 69 ], which demonstrate the chloroquine effect on P . vivax .…”

Section: Discussionsupporting

confidence: 91%

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi

et al. 2018

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BackgroundThe mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America.MethodsTo determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 μg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated.ResultsIVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions.ConclusionIn conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

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Section: Discussionsupporting

confidence: 91%

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi

et al. 2018

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“…T cell surface were stained with anti-CD4, anti-CD8, anti-CD3, anti-CD69, anti-HLA-DR, anti-Ki67, anti-CD19, anti-γδ TCR, anti-αβ TCR, anti-CD56, anti-CD161 and anti-TCRVα7.2. RBCs were stained with anti-CD71, anti-CD235a, anti-HLA-ABC (class I), anti-HLA-DR (class II), Thiazole Orange and SYBR Green I ( P. vivax DNA) 25,42–44 . To analyze intracellular cytokine and granule protein expression, cells were incubated at 37°C, 5% CO 2 in the presence of indicated stimuli for 30 min before adding Brefeldin A (1μl/ml) and Monensin (1μl/ml) (BD Pharmingen) solutions and culture for an additional 4–10 hr prior to staining.…”

Section: Methodsmentioning

confidence: 99%

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax–infected reticulocytes

Junqueira

Barbosa

Costa

et al. 2018

Nat Med

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Plasmodium vivax causes approximately 100 million clinical malaria cases yearly. The basis of protective immunity is poorly understood and thought to be mediated by antibodies. Cytotoxic CD8 T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8 T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8 T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8 T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

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“…Various types of permeable markers can be used to mark the DNA [ 85 ]. The mixture of infected and non-infected red blood cells is then analysed in a cytometer, and the results analysed to determine the amount of infected cells, hence the number of parasites having grown in absence or in presence of the anti-malarial drug, to determine the IC 50 [ 86 , 87 ].…”

Section: In Vitro and Ex Vivo Phenotypic Assay For Anti-malarial Suscmentioning

confidence: 99%

Tools for surveillance of anti-malarial drug resistance: an assessment of the current landscape

Nsanzabana

Djallé

Guérin

et al. 2018

Malar J

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To limit the spread and impact of anti-malarial drug resistance and react accordingly, surveillance systems able to detect and track in real-time its emergence and spread need to be strengthened or in some places established. Currently, surveillance of anti-malarial drug resistance is done by any of three approaches: (1) in vivo studies to assess the efficacy of drugs in patients; (2) in vitro/ex vivo studies to evaluate parasite susceptibility to the drugs; and/or (3) molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance. These methods are complementary, as they evaluate different aspects of resistance; however, standardization of methods, especially for in vitro/ex vivo and molecular techniques, is lacking. The World Health Organization has developed a standard protocol for evaluating the efficacy of anti-malarial drugs, which is used by National Malaria Control Programmes to conduct their therapeutic efficacy studies. Regional networks, such as the East African Network for Monitoring Antimalarial Treatment and the Amazon Network for the Surveillance of Antimalarial Drug Resistance, have been set up to strengthen regional capacities for monitoring anti-malarial drug resistance. The Worldwide Antimalarial Resistance Network has been established to collate and provide global spatial and temporal trends information on the efficacy of anti-malarial drugs and resistance. While exchange of information across endemic countries is essential for monitoring anti-malarial resistance, sustainable funding for the surveillance and networking activities remains challenging. The technology landscape for molecular assays is progressing quite rapidly, and easy-to-use and affordable new techniques are becoming available. They also offer the advantage of high throughput analysis from a simple blood spots obtained from a finger prick. New technologies combined with the strengthening of national reference laboratories in malaria-endemic countries through standardized protocols and training plus the availability of a proficiency testing programme, would contribute to the improvement and sustainability of anti-malarial resistance surveillance networks worldwide.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2185-9) contains supplementary material, which is available to authorized users.

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Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry

Cited by 16 publications

References 38 publications

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax–infected reticulocytes

Tools for surveillance of anti-malarial drug resistance: an assessment of the current landscape

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