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

Roobsoong W, Tharinjaroen CS, Rachaphaew N, Chobson P, Schofield L, Cui L, Adams JH, Sattabongkot J - Malar. J. (2015)

Bottom Line: 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.Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low.

View Article: PubMed Central - PubMed

Affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. wanlapa.ros@mahidol.edu.

ABSTRACT

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% O2, 5% CO2, and 90% N2). 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.

No MeSH data available.


Related in: MedlinePlus

Parasite density of Plasmodium vivax cultured with different sources of reticulocytes in 7 days. a Parasite density during the 7 day cultured period; b Magnification of the graph from day 4 to day 7. Reticulocytes purified from peripheral blood (RP), cord blood (RC) and haematopoietic stem cell (cRBC) were used to cultured fresh isolates of P. vivax (10 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. Each box represents average parasite density from 7 days obtained from 10 P. vivax isolates.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4524445&req=5

Fig3: Parasite density of Plasmodium vivax cultured with different sources of reticulocytes in 7 days. a Parasite density during the 7 day cultured period; b Magnification of the graph from day 4 to day 7. Reticulocytes purified from peripheral blood (RP), cord blood (RC) and haematopoietic stem cell (cRBC) were used to cultured fresh isolates of P. vivax (10 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. Each box represents average parasite density from 7 days obtained from 10 P. vivax isolates.

Mentions: The influence of sources of reticulocytes to the invasion and maturation of vivax parasites was determined. For comparison, reticulocytes were obtained from two additional sources: purified from peripheral blood (RP), and cultured from haematopoietic stem cells (cRBC). All reticulocytes were seeded at 4% and cultured with the same clinical isolates of P. vivax. Invasion and maturation of the parasites in these cultures were monitored at 18 and 48 h, respectively. The published criteria for developmental assessment of P. vivax was used to differential stage of the parasite [2]. Ring-stage parasitaemias were not significantly (one way ANOVA, F = 0.192, P = 0.827) different among the three sources of reticulocytes. In addition, the morphology of the parasites residing in RP, RC and cRBC at 18 h did not show obvious differences (Fig. 2a). However, at 48 h parasites in RP showed better maturation than those in RC and cRBC (Fig. 2b). Nonetheless, parasite growth in all three sources of reticulocytes was slightly delayed and none of the parasite isolates tested could complete blood stage schizogony within 48 h. Further, the long-term effect of different sources of reticulocytes on parasite culture was studied with 11 P. vivax clinical isolates (Fig. 3). Reticulocytes were added daily to maintain 4% and the parasite density was monitored daily for 7 days. Overall, parasite densities among the three reticulocyte sources were not significantly different (one way ANOVA, F value = 0.024, P = 0.976). Yet, depending on the parasite isolates, some grew better in one source of reticulocytes than others (see Additional file 1). Although RC reticulocytes contain fetal haemoglobin, which was reported to have an inhibitory effect on parasite growth [13], RC better supported parasite maturation as well as gametocyte production in four of the tested parasite isolates (see Additional file 2). Given that RP could support parasite growth for most of the tested isolates, and are a more reliable source of reticulocytes, RP reticulocytes were chosen for subsequent studies to improve long-term culture of P. vivax.Fig. 2


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

Roobsoong W, Tharinjaroen CS, Rachaphaew N, Chobson P, Schofield L, Cui L, Adams JH, Sattabongkot J - Malar. J. (2015)

Parasite density of Plasmodium vivax cultured with different sources of reticulocytes in 7 days. a Parasite density during the 7 day cultured period; b Magnification of the graph from day 4 to day 7. Reticulocytes purified from peripheral blood (RP), cord blood (RC) and haematopoietic stem cell (cRBC) were used to cultured fresh isolates of P. vivax (10 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. Each box represents average parasite density from 7 days obtained from 10 P. vivax isolates.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524445&req=5

Fig3: Parasite density of Plasmodium vivax cultured with different sources of reticulocytes in 7 days. a Parasite density during the 7 day cultured period; b Magnification of the graph from day 4 to day 7. Reticulocytes purified from peripheral blood (RP), cord blood (RC) and haematopoietic stem cell (cRBC) were used to cultured fresh isolates of P. vivax (10 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. Each box represents average parasite density from 7 days obtained from 10 P. vivax isolates.
Mentions: The influence of sources of reticulocytes to the invasion and maturation of vivax parasites was determined. For comparison, reticulocytes were obtained from two additional sources: purified from peripheral blood (RP), and cultured from haematopoietic stem cells (cRBC). All reticulocytes were seeded at 4% and cultured with the same clinical isolates of P. vivax. Invasion and maturation of the parasites in these cultures were monitored at 18 and 48 h, respectively. The published criteria for developmental assessment of P. vivax was used to differential stage of the parasite [2]. Ring-stage parasitaemias were not significantly (one way ANOVA, F = 0.192, P = 0.827) different among the three sources of reticulocytes. In addition, the morphology of the parasites residing in RP, RC and cRBC at 18 h did not show obvious differences (Fig. 2a). However, at 48 h parasites in RP showed better maturation than those in RC and cRBC (Fig. 2b). Nonetheless, parasite growth in all three sources of reticulocytes was slightly delayed and none of the parasite isolates tested could complete blood stage schizogony within 48 h. Further, the long-term effect of different sources of reticulocytes on parasite culture was studied with 11 P. vivax clinical isolates (Fig. 3). Reticulocytes were added daily to maintain 4% and the parasite density was monitored daily for 7 days. Overall, parasite densities among the three reticulocyte sources were not significantly different (one way ANOVA, F value = 0.024, P = 0.976). Yet, depending on the parasite isolates, some grew better in one source of reticulocytes than others (see Additional file 1). Although RC reticulocytes contain fetal haemoglobin, which was reported to have an inhibitory effect on parasite growth [13], RC better supported parasite maturation as well as gametocyte production in four of the tested parasite isolates (see Additional file 2). Given that RP could support parasite growth for most of the tested isolates, and are a more reliable source of reticulocytes, RP reticulocytes were chosen for subsequent studies to improve long-term culture of P. vivax.Fig. 2

Bottom Line: 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.Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low.

View Article: PubMed Central - PubMed

Affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. wanlapa.ros@mahidol.edu.

ABSTRACT

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% O2, 5% CO2, and 90% N2). 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.

No MeSH data available.


Related in: MedlinePlus