This study reports the progress made toward long-term in vitro culture of
P. vivax isolates originating from infected patients in Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. This region has previously been shown to experience consistent transmission of
P. vivax [
63,
72,
87], including among Duffy-negative people [
63]. Important first steps for understanding the genetic characteristics and biology of
P. vivax parasites from this region require developing reliable methods for collecting, storing, transporting field-collected blood samples, and culturing parasites from those samples. All of the long-term in vitro culture work performed here has used whole blood from squirrel monkeys (
S. boliviensis), without reticulocyte enrichment. These methods have reproducibly yielded long-term cultures of all four
P. vivax isolates attempted so far (median time 159 days, range 36–233 days); none of the isolates failed to propagate using this culture system. This reproducibility was observed regardless of the anticoagulant, K
+-EDTA or Na
+-heparin, used in vacutainers to collect and store patient blood samples before cryopreservation. Furthermore, one of these isolates, AMP2014.01, demonstrated reproducible capacity for culture in Saimiri RBCs (two different long-term cultures have been initiated from separate cryopreserved blood sample vials), retained capacity for infecting human RBCs, and could be cryopreserved and re-cultivated.
Cultivation of Plasmodium vivax patient isolates
The four P. vivax isolates cultured in this study were collected from patients with parasitaemias as low as 0.024% to as high as 0.27%. In addition, the time interval between sample collection and in vitro culture varied from 166 to 961 days. Despite this variation, a final pellet of intact RBCs between 30 to 80 μL was regularly observed when these patient samples were thawed and prepared for in vitro culture. The starting culture parasitaemias on day 0 from these thawed samples are likely to have been lower than the corresponding patient parasitaemias due to lysis during the thawing process. Thus, these methods proved to be efficient and reproducible in initiating cultures from small amounts of parasite material, and the success of culturing did not appear to be limited by low patient parasitaemia.
Along this line that these cultivation methods were efficient and reproducible in initiating cultures from low parasite material, cryopreservation and re-cultivation of the cultured parasites was achieved successfully. Seventy-five microliter of isolate AMP2014.01 (second culture) at a parasitaemia of 0.35% were cryopreserved. When this aliquot was thawed, a 20 μL pellet of intact RBCs was recovered and a culture in one 1-mL well was initiated. From this starting material, a parasitaemia of 0.4% was observed on day 33; the culture was split into two 1-mL wells on day 35, and was terminated on day 88.
An overall assessment of the growth dynamics of the patient isolates AMP2014.01 (first culture, 233 days; second culture, 165 days), AMP2014.02 (155 days), and AMP2016.02 (159 days) shows that the parasitaemia generally ranged 0.1–0.4%; for isolate AMP2014.02, it reached 1% twice during the culture period. A unique growth pattern was observed for isolate AMP2014.01 (first culture), where the parasitaemia fluctuated considerably during the first 60 days, and then maintained for the remaining culture period. This observation in particular is similar to those made by Roobsoong et al. [
50] regarding patient isolates cultured in modified McCoy’s 5A medium with reticulocytes purified from adult peripheral blood added daily. In their culture system, parasite density was dramatically dropped during the first week of culture and then maintained at very low density for the whole culture period. Most of their 30 isolates shared a similar growth pattern with fluctuations of parasitaemia (Fig. 7b [
50]).
As lysis (~ 50%) was observed in the culture every 96–120 h, Saimiri RBCs were added to maintain 4% haematocrit. The lysis did not appear to reflect expansion of the culture. Extensive breakage of Saimiri erythrocytes in the culture was reported previously by Lanners [
45], which may reflect the extensive host-cell modifications that the parasite induces in the infected erythrocytes [
88].
It is important to note that microscopic evaluation of the cultures was performed by counting a total of 1000 RBCs (10–20 100× fields, 50–100 RBCs/field) from 2 μL of resuspended cultures. Also, the intervals when the slides were made to evaluate parasitaemia were not regular, and ranged from 2 to 15 days. Average daily rate of change in parasitaemia of isolate AMP2014.01 (first culture) over 233 days culture period, divided into early (days 0–60, 10 observations) and later (days 75–233, 20 observations) periods, was estimated. Linear regression analysis showed that, with a patient parasitaemia of 0.13% at the initiation of the culture (day 0), the parasitaemia significantly increased by 0.064% (95% confidence interval 0.032–0.095, P = 0.001) per day during the early period. However, with parasitaemia reaching 0.3% on day 75, the parasitaemia did not significantly change during the later period. The growth dynamics of isolates AMP2014.01 (second culture) and AMP2016.02 were highly similar to that of isolate AMP2014.01 (first culture) during the later period. It is possible that counting more RBCs per slide and/or making slides to evaluate parasitaemia at a regular interval, every 48 h cycle of invasion and multiplication, may have provided a more accurate assessment of parasite growth dynamics and multiplication rate.
Finally, molecular characterization of the PvDBP and PvAMA-1 segments was performed, which were amplified from all four patient isolates and AMP2014.01 (first culture) aliquots taken on days 86 and 202. This analysis provides some assessment of the genetic diversity in these samples, shows differences from the commonly used parasite strains, and proves that the AMP2014.01 patient isolate and culture aliquots carry the same haplotypes. Efforts to generate enough cultured packed cell volume required to carry out further genome-wide analysis are underway.
Cross-species propagation
Given that long-term in vitro culture of
P. vivax has been possible in RBCs from
S. boliviensis, a question was whether this cross-species exposure eliminated the ability of
P. vivax to invade human RBCs. The experimental results summarized in Fig.
3d–f, indicate that isolate AMP2014.01 retains the ability to invade human RBCs in the context of short-term invasion assays. Results that
P. vivax strains exposed to Saimiri and Aotus RBCs do not lose their abilities to infect human RBCs are not unexpected given previously published results. Since the late 1980s, multiple monkey-adapted
P. vivax strains (including Belem [
41], Chesson [
44], AMRU-1 [
89] and Sal-1 [
56]) have successfully been used under a number of different study designs to infect human RBCs.
While it is encouraging that Saimiri-adapted parasites were still able to invade human RBCs after 25 days in culture, it is acknowledged that this is a short time period. It remains to perform such studies using parasites which have been in culture for a longer period, and to investigate the variability of successful human RBC invasion among different patient isolates.
Comparisons with previous approaches
Other groups have performed numerous well-designed investigations to improve in vitro culture of
P. vivax. Given the robust productivity experienced with in vitro cultivation of the Malagasy
P. vivax isolates in Saimiri RBCs, this system was compared to previous studies (Additional file
3: Table S2). Most of the previous studies have focused their efforts on long-term and short-term in vitro culture of
P. vivax in human RBCs, with the notable exception of Mons et al. [
90], who used Aotus RBCs for short-term growth of the parasite.
One previous study, by Lanners, most similar to the work performed here, attempted prolonged in vitro culture of the
P. vivax Chesson strain in Saimiri RBCs and in a 1:1 mixture of Saimiri and human RBCs [
45]. Further comparison with Lanners provides a contrast in methods and outcomes compared to the present study. In the discussion of his study, which concluded by sustaining low levels of
P. vivax in vitro propagation for 16–22 days, Lanners commented that attempts to culture the parasite in Saimiri RBCs and Saimiri serum were abandoned owing to the fragility of the monkey RBCs. His continued ability to maintain his cultures was the result of providing human reticulocytes and 15% human AB serum. Lanners’ work and many other earlier studies used RPMI 1640 as the base of their culture medium. Maintaining
P. vivax laboratory as well as Malagasy patient isolate cultures using RPMI 1640 has been very difficult; our those efforts were marked by inconsistent propagation and early termination of cultures (Grimberg and Zimmerman, unpublished observations). Methods from other recent studies have also shown evidence of moving away from this medium (Additional file
3: Table S2). In addition to the use of AIM V medium, the cultures in the present study were supplemented with a lower concentration (10%) of heat-inactivated human AB serum than most other studies (range from 10 to 50%; Additional file
3: Table S2). Finally, the gas mixture used was 10% CO
2. AIM V medium formulation is proprietary and has not been disclosed by the manufacturer. However, as mentioned previously, the base medium for AIM V is DMEM (manufacturer’s notes). Since the content of NAHCO
3 in the culture medium determines the CO
2 concentration for culturing, 10% CO
2 was used in the present study (NAHCO
3 content in DMEM, 3.7 g/L). In comparison, the NAHCO
3 content of RPMI 1640 is 2.0 g/L, therefore 5% CO
2 is used.
The preference
P. vivax displays for reticulocytes has been demonstrated by both in vivo [
52,
54,
55] and in vitro observations [
41,
50,
51,
55,
56]. Acknowledging the challenges of
P. vivax in vitro culture [
40‐
49], together with previous experience [
91], reticulocyte enrichment was investigated. Those efforts included strategies that preferentially lyse older RBCs [
92], as well as strategies that separate reticulocytes from older RBCs by centrifugation through Percoll or selection by CD71 positivity [
47,
51]. Overall, these methods have produced variable results. The in vitro cultivation method evaluated here has not included reticulocyte enrichment. One reason for not including reticulocyte enrichment was the small volume (2–3 mL [maximum]) of Saimiri blood requested every 2 weeks. Studies that have performed reticulocyte enrichment to culture
P. vivax have used larger volumes of blood [
44,
50,
56]. Interestingly, the lack of reticulocyte enrichment has not appeared to hamper adaptation of the Malagasy
P. vivax isolates to in vitro propagation in Saimiri blood. Saimiri blood contains about 2% reticulocytes [
85]. However, it is believed that reticulocyte counts in Saimiri blood, stored at 4 °C, are not available. In the present study, in the leukocyte-depleted Saimiri blood preparations, stored at 4 °C, reticulocyte counts were stable at about 2% over a 2-week period, which is when the fresh blood was obtained. This is in accordance with the results of a study on leukocyte-depleted human blood, stored for 6 weeks under standard blood bank conditions (2–6 °C) [
93]. In this study, reticulocyte counts increased at day 21 and remained constant over time. Finally, recent reports have demonstrated a preference of the parasite for young reticulocytes, including that
P. vivax Sal-1 rings ex vivo were found in young reticulocytes [
56]. However, in this same study, the second generation (20 h in vitro) rings and trophozoites were found in older reticulocytes and mature RBCs [
56]. Whether the storage of Saimiri blood, containing reticulocytes, for 2–3 weeks affects the invasion and maturation of the
P. vivax isolates remains to be explored. For now, avoiding reticulocyte enrichment has saved considerable time and resources, and appears to simplify maintenance of
P. vivax cultures in the present system.
Limitations and future directions
Despite the experience of maintaining in vitro culture of multiple P. vivax isolates from Madagascar in Saimiri RBCs, the focus here on isolate AMP2014.01 encounters some limitations. The authors recognize that they have not performed the same manipulations demonstrated for AMP2014.01 with all of the isolates introduced in this study. Similarly, studying the receptivity of different P. vivax isolates to cryopreservation and onward rounds of in vitro culture will need to be performed. Such studies would further substantiate if the approaches described here are able to successfully contribute to the development of stable parasite strains that are characterized by distinct biological phenotypes from Madagascar and other endemic areas.
Beyond demonstrating the basic capacity of
P. vivax in vitro culture in Saimiri RBCs, it will be important to investigate further if this system can be used to characterize the specific factors involved in
P. vivax RBC invasion, as has been performed by laboratory-adapted strains of
P. falciparum [
33,
94,
95]. Therefore, a number of in vitro studies are readily anticipated. These include tests to determine if treatments to manipulate the RBC surface (e.g., trypsin, chymotrypsin, neuraminidase) and antibody reagents interacting with the Duffy blood group and Duffy binding protein affect parasite RBC invasion in predictable ways. Beyond this basic assessment of
P. vivax in an in vitro setting, it will be exciting to determine if the methods described here will facilitate a wider range of investigations into the lifecycle of this parasite to include attaining gametocytes, exposure of mosquito hosts to different
P. vivax strains, evaluation of liver-stage infection and development of hypnozoites. These studies, as well as analyses of gene expression and genetic manipulation of this parasite would all be facilitated if it is possible to develop stable, laboratory-adapted strains of
P. vivax. Finally, investigation of the possible differences in expression/biology of the parasite grown in Saimiri
vs. human RBCs would also be facilitated.