The goal of malaria eradication 
cannot be realized without adequate interventions to eliminate infections by all species of malaria parasites that infect humans. Pv causes much less severe disease and death than does P. falciparum
, but is more widespread globally and infects 80–300 million people every year 
. Unlike the other major human malaria parasite, P. falciparum
, there is no in vitro
cultivation system to produce Pv asexual and sexual erythrocytic stage parasites. Also, like P. falciparum
there is no small animal model available for Pv. These deficiencies undermine efforts to develop drugs and vaccines against Pv, especially against the pre-erythrocytic stages. Elimination of Pv would be facilitated by development of an easily administered drug that eliminates all liver stage parasites, including hypnozoites, and can be given to the entire population in mass treatment campaigns without concern for life threatening side effects. Identification and development of such a drug would be facilitated by an in vitro
Herein, we report the first steps toward development of a medium throughput and high content assay to screen for new drugs against Pv liver stages. One of the serious problems with developing such an assay is that it has been extremely difficult to acquire Pv spz for such studies, and that each time a study is done, the study has to be done with spz from a different infection and/or source. This of course limits the capacity to do multiple, reliable comparisons. For the first time we report that cryopreserved Pv spz from the same lot can be used to reproducibly infect hepatoma cells, and that the numbers of 3 day hepatocyte stage parasites are similar in cultures infected with fresh and cryopreserved Pv spz (). Similar results have now been obtained with cryopreserved Pv spz in the 9 day assay (Velmurugan, S, manuscript in preparation). Being able to produce and vial large single lots of Pv spz that can be used in multiple experiments over time is a first prerequisite to developing a medium to high throughput screening assay.
The next step is to make the assay reproducibly quantitative. The earlier reports on culturing Pv liver stages 
provided primarily qualitative data that described the ability of primary human hepatocytes or human hepatocyte lines to support the development of hepatocyte stages of Pv and the morphology of these parasites. In the present study, our goal was to measure the rate of infectivity of Pv spz in HepG2-A16 cells and rate of development of invaded Pv spz into hepatocyte stages. We observed that when 25,000 Pv spz were added to 20,000 HepG2-A16 cells and the culture was maintained for 3 days, the overall rate of infectivity was 1.8%, and this was reduced by about 50% to 0.9% when the cultures were maintained for 9 days (). Our data with Pf spz cultured in HC-04 cells (Chakravarty, S, unpublished), as well as Pv spz cultured in HepG2 cells (Velmurugan, S, unpublished) indicate that about 50% of the spz added to the wells at the outset of the culture are washed out 3 hours later. Thus, of the spz remaining, the overall infectivity is double that which we record for both the 3 and 9 day assays. We think that the difference in numbers of parasites between 3 and 9 day assays is attributable to 1) the fact that some of the 3 day parasites do not fully develop, 2) hepatoma cell death during the period from 3 to 9 days, including cells containing parasites, and 3) replication of the uninfected hepatoma cells in the wells, which makes it more difficult to visualize parasites after 9 days. The infectivity of Pv spz in the HC-04 human hepatocyte line is reported to be 0.041% 
. HepG2-A16 and HC-04 hepatocyte lines differ biologically, but both of them support development of hepatocyte stages of Pv. The difference in the results from Thailand with HC-04 cells 
and our results in regard to rate of infectivity in these two lines could be due to the inherent capacity for infection of the two cell lines. However, other factors could have been responsible for the different infectivity rates. We used a single strain (India VII), which has been maintained at CDC since 2001 
and fed mosquitoes on infected chimpanzee blood. The Thai group used Pv spz produced from gametocytes infecting people in the field. Another potential factor may be that the purified Pv spz that we use may be associated with higher infection rates, as compared to the unpurified Pv spz used by all other groups. In unpurified Pv spz preparations there may be contaminant molecules of mosquito origin, which compete with Pv spz to bind to the hepatocytes, but purification of Pv spz from these mosquito components might allow Pv spz to attach to and invade hepatocytes more efficiently and at higher rate.
Next we showed, for the first time, that our hepatocyte stage culture system could be used to assess drugs against Pv liver stages in vitro. There was a clear dose response when using PQ ( and ). Most data suggest that the primary activity in vivo when PQ is used to eliminate infected hepatocytes is by a metabolite of primaquine, not by the parent compound. This is probably the reason why such large quantities of primaquine (EC50 of 0.1 to 1.0 µg/mL) were required, even for the 9-day assay. Work is in progress in an attempt to isolate the active metabolites of primaquine (Colin Ohrt, personal communication) and we anticipate assessing its activity in the future.
Dose dependent effect of primaquine on the development of 3 day hepatocyte stage parasites.
Dose dependent effect of primaquine on the development of 9 day hepatocyte stage parasites.
In the 9-day assay 10 µg/mL inhibited parasite development by 86% while in the 3-day assay there was only a 36% reduction at 10 µg/mL. The total cumulative dose of PQ is important for complete elimination of liver stages of Pv in vivo 
. The greater effect of PQ observed in the 9-day assay as compared to the 3-day assay may have been due to the cumulative effect of PQ over 9 days as compared to only 3 days. Future studies will assess the kinetics of activity of PQ and other drugs by looking at the results after 3 to 9 days of treatment. At 10 µg/mL for 9 days, hepatotoxicity was observed, as has been seen in cultures of P. cynomolgi
and P. knowlesi
hepatocyte stages in primary rhesus monkey hepatocytes 
. This would be a limiting variable in these cultures. However, subsequent work with a different lot of primaquine has shown much less cytotoxicity (Velmurugan, S, unpublished).
An ideal drug for elimination of Pv will be effective against hypnozoites. Hypnozoites or dormant hepatocyte stages of Pv were observed in vivo
and possibly in HepG2-A16 cells after infection with Pv spz 
. Identifying and characterizing hypnozoites is a major emphasis of our research program. Thus, the small forms expressing PvCSP that we have identified in the 9-day cultures intrigue us. Much work will be needed to determine if these are truly hypnozoites.
We have established the capacity to reproducibly infect hepatoma cells with Pv spz, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. We are now moving to a 96-well format that will use less Pv spz/well and less time to quantitate infected hepatocytes, automated read outs, establishment of the effect of other drugs on the cultures and characterization of the small forms. With these improvements, we anticipate a medium throughput assay can be developed in near future that can be used to screen for new drugs against Pv hepatocyte stages.