Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax

Background: The study of the biology, transmission and pathogenesis of Plasmodium vivax is hindered due to the lack of a robustly propagating, continuous culture of this parasite. The current culture system for P. vivax parasites still suffered from consistency and difficulties in long-term maintenance of parasites in culture and for providing sufficient biological materials for studying parasite biology. Therefore, further improvement of culture conditions for P. vivax is needed. Methods: Clinical samples were collected from patients diagnosed with P. vivax in western Thailand. Leukocyte-depleted P. vivax infected blood samples were cultured in a modified McCoy's 5A medium at 5% haematocrit under hypoxic condition (5% O 2 , 5% CO 2 , and 90% N 2 ). Reticulocytes purified from adult peripheral blood were added daily to maintain 4% reticulocytes. Parasites were detected by microscopic examination of Giemsa-stained smears and molecular methods. Results: The effects of culture variables were first analysed in order to improve the culture conditions for P. vivax. Through analysis of the sources of host reticulocytes and nutrients of culture medium, the culture conditions better supporting in vitro growth and maturation of the parasites were identified. Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low. Conclusions: Based on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed

Aditional file 1.

Parasite density of P. vivax cultured with different sources of reticulocytes in 7 days. Reticulocytes purified from peripheral blood (red line), cord blood (blue line) and hematopoietic stem cell (green line) were used to cultured fresh isolates of P. vivax (11 isolates). Reticulocytes were added to the cultures daily at a final 4% and the parasite density was determined for 7 days from Giemsa-stained thick smears (1 Âľl packed cells). Each line represents average parasite density from 7 days obtained from 11 P. vivax isolates

Aditional file 2.

Gametocyte density of P. vivax cultured with different sources of reticulocytes in 7 days. Reticulocytes purified from peripheral blood (red line), cord blood (blue line) and hematopoietic stem cell (green line) were used to cultured fresh isolates of P. vivax (11 isolates). Reticulocytes were added to the cultures daily at a final 4% and the gametocyte density was determined for 7 days from Giemsa-stained thick smears (1 Âľl packed cells). Each line represents average parasite density from 7 days obtained from 11 P. vivax isolates. Cord blood-reticulocytes has highly expression of fetal hemoglobin but still shown better support gametocyte production in some parasite isolates (VKTS33, VKBT59, VKBT63, and VKBT81)

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus

International audiencePlasmodium vivax is now the predominant species causing malarial infection and disease in most non-African areas, but little is known about its transmission efficiency from human to mosquitoes. Because the majority of Plasmodium infections in endemic areas are low density and asymptomatic, it is important to evaluate how well these infections transmit. Using membrane feeding apparatus, Anopheles dirus were fed with blood samples from 94 individuals who had natural P. vivax infections with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate was positively correlated with blood parasitemia and that infection began to rise when parasitemia was >10 parasites/ll. Below this threshold, mosquito infection is rare and associated with very few oocysts. These findings provide useful information for assessing the human reservoir of transmission and for establishing diagnostic sensitivity required to identify individuals who are most infective to mosquitoes

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies