Effects of Artesunate on Parasite Recrudescence and Dormancy in the Rodent Malaria Model Plasmodium vinckei

Artemisinin (ART) is the recommended first line therapy for treating uncomplicated and drug-resistant Plasmodium falciparum, the most pathogenic form of malaria. However, treatment failure following ART monotherapy is not uncommon and resistance to this rapidly acting drug has been reported in the Thai-Cambodian border. Recent in vitro studies have shown that following treatment with dihydroartemisinin (DHA), the development of ring-stage parasites is arrested for up to 20 days. These arrested (i.e. dormant) rings could be responsible for the recrudescence of infection that is observed following ART monotherapy. To develop a better understanding of the stage-specific effects of ART and determine if dormancy occurs in vivo, the ART derivative artesunate (AS) was used to treat mice infected with the synchronous rodent malaria parasites P. vinckei petteri (non-lethal) and P. v. vinckei (lethal). Results show that in both the non-lethal and lethal strains, ring-stage parasites are the least susceptible to treatment with AS and that the day of treatment has more of an impact on recrudescence than the total dose administered. Additionally, 24 hrs post-treatment with AS, dormant forms similar in morphology to those seen in vitro were observed. Finally, rate of recrudescence studies suggest that there is a positive correlation between the number of dormant parasites present and when recrudescence occurs in the vertebrate host. Collectively, these data suggest that dormancy occurs in vivo and contributes to recrudescence that is observed following AS treatment. It is possible that this may represent a novel mechanism of parasite survival following treatment with AS

Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development.</p> <p>Methods</p> <p>The <it>P. falciparum </it>gene, <it>PF11_0394</it>, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR) and green fluorescent protein (GFP)-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined.</p> <p>Results</p> <p>The PF11_0394 protein has orthologs in other <it>Plasmodium </it>species and Apicomplexans, but none outside of the group Apicomplexa. <it>PF11_0394 </it>transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites.</p> <p>Conclusions</p> <p>PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in <it>P. falciparum </it>biology, it could be another candidate for a new vaccine.</p

Quinolone-based compounds, formulations, and uses thereof

Provided herein are quinolone-based compounds that can be used for treatment and/or prevention of malaria and formulations thereof. Also provided herein are methods of treating and/or preventing malaria in a subject by administering a quinolone-based compound or formulation thereof provided herein

Survival and time to recrudescence following inoculation of artesunate-treated <i>P. v. vinckei</i> parasites.

<p>There were a total of 16 experimental groups with five mice per group (n = 5). Untreated <i>P. v vinckei</i> parasites were injected into experimental mice using quantities from the lowest and highest dose in the dormant parasite dilution series.</p>a<p>Means significantly different to untreated 4×10⁶, <i>p</i><0.05; Dunnett's multiple comparison test. N/A = never reached greater than 5%. Data are means ±SD.</p

<i>P. v. vinckei</i> morphology in mice before treatment and 24 hr following treatment with artesunate.

<p>A. Two representative images are shown for each stage of development <i>in vivo</i>. Ring-stage (R), young trophozoite (Y-troph), mid-term trophozoite (M-troph), old trophozoite (O-troph), and schizont. (SCH). B. Treated ring-stage parasites (top row) have a condensed nucleus and pyknotic appearance. Older parasite stages treated with artesunate (bottom row) appear to have an absence of hemozoin and loss of membrane integrity.</p

The effects of repeated dosing with AS on <i>P. v. vinckei</i>-infected mice.

<p>Mice were treated with a total dose of 256 mg/kg of AS and monitored for recrudescence (n = 5 mice per group). A. Mice treated for three or four consecutive days had the highest percent survival (70%) when compared to the untreated (Dunnett's multiple comparison test, <i>p</i><0.01). B. In the variable group, mice treated on days 3 and 7 post-infection (PI) had the highest percent survival (80%) when compared to the untreated (Dunnett's multiple comparison test, <i>p</i><0.05).</p

Experimental survival rates using <i>P. v. vinckei (Pvv)</i>, a lethal strain, compared to peak parasitemias from a previous study using <i>P. v. petteri (Pvp)</i>, a non-lethal strain.

<p>There were five mice per group in the lethal and non-lethal pilot studies. The most effective regimens in comparison to the untreated group are in bold.</p>*<p> = <i>p</i><0.05; Dunnett's multiple comparison test; N/A = not applicable.</p

The stage-specific effects of a single dose of AS.

<p>A. <i>Plasmodium vinckei petteri</i>-infected mice (n = 8 mice per group) were treated with one dose of AS (64 mg/kg). Comparison of peak parasitemias revealed that all treated groups had significantly lower peak parasitemias when compared to the untreated control (Dunnett's multiple comparison test, <i>p</i><0.001). * = statistically significant difference. B. Assessment of average daily parasitemias showed that the ring stage had the highest average parasitemia when compared to treated trophozoites and schizonts. Both the ring and trophozoite groups had a secondary peak on days 27 and 14 PI, respectively. Overall, the treated trophozoites took the longest to clear and had the lowest average parasitemia. Data are means ± SD. C. Mice infected with the lethal strain <i>P. v. vinckei</i> (n = 5 mice per group) were treated with one dose of AS (64 mg/kg). The median day of survival for untreated mice, and mice treated when mostly rings, trophozoites, or schizonts were present was 7.0 and 9.0, respectively. No significant difference in survival was observed among the treated groups.</p

Experimental design for the stage-specific studies.

<p>Donor mice were used to infect one batch of 25 or 40 experimental mice with either <i>P. vinckei vinckei or P. vinckei petteri</i>, respectively. Mice then were randomly separated into groups of 5 consisting of untreated, artesunate treated (AS) rings, trophozoites (trophs), and schizonts. Amodiaquine treated parasites were included as a positive drug control. Parasitemia were monitored for 30 days.</p

Primary stages present during dormant parasite recrudescence in intact mice.

<p>The average parasitemia versus time in relation to the number of dormant parasites are shown in the figure (n = 5 mice per group). Rate of recrudescence from dormancy was defined as the average time to >5.0% parasitemia. In all treatment groups, with the exception of the untreated 4×10⁶, rings are the predominant stage present as parasites begin to recover. A (untreated 4×10⁶), B (untreated 4×10¹), C (dorm 4×10⁶), D (dorm 4×10⁵), E (dorm 4×10⁴), F (dorm 4×10³), G (dorm 4×10²) andH (dorm 4×10¹). Ring (R), young trophozoite (YT), mid-term trophozoite (MT), old trophozoite (OT), and schizont (SCH).</p