RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

The recent identification of antisense RNA in the transcriptomes of many eukaryotes has generated enormous interest. The presence of antisense RNA in Plasmodium falciparum, the causative agent of severe malaria, remains controversial. Elucidation of the mechanism of antisense RNA in P. falciparum synthesis is critical in order to demonstrate the origin and function of these transcripts. Therefore, a systematic analysis of antisense and sense RNA synthesis was performed using direct labeling experiments. Nuclear run on experiments with single-stranded DNA probes demonstrated that antisense RNA is synthesized in the nucleus at several genomic loci. Antisense RNA synthesis is sensitive to the potent RNA polymerase II inhibitor α-amanitin. Antisense and sense transcription was also detected in nuclei isolated from synchronized parasites, suggesting concurrent synthesis. In summary, our experiments directly demonstrate that antisense RNA synthesis is a common transcriptional phenomenon in P. falciparum, and is catalyzed by RNA polymerase II. Copyright © 2005 RNA Society

Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres

BACKGROUND: Controlled human malaria infection (CHMI) accelerates development of anti-malarial interventions. So far, CHMI is done by exposure of volunteers to bites of five mosquitoes carrying Plasmodium falciparum sporozoites (PfSPZ), a technique available in only a few centres worldwide. Mosquito-mediated CHMI is logistically complex, exact PfSPZ dosage is impossible and live mosquito-based interventions are not suitable for further clinical development. METHODS: An open-labelled, randomized, dose-finding study in 18-45 year old, healthy, malaria-naive volunteers was performed to assess if intravenous (IV) injection of 50 to 3,200 aseptic, purified, cryopreserved PfSPZ is safe and achieves infection kinetics comparable to published data of mosquito-mediated CHMI. An independent study site verified the fully infectious dose using direct venous inoculation of PfSPZ. Parasite kinetics were assessed by thick blood smear microscopy and quantitative real time PCR. RESULTS: IV inoculation with 50, 200, 800, or 3,200 PfSPZ led to parasitaemia in 1/3, 1/3, 7/9, and 9/9 volunteers, respectively. The geometric mean pre-patent period (GMPPP) was 11.2 days (range 10.5-12.5) in the 3,200 PfSPZ IV group. Subsequently, six volunteers received 3,200 PfSPZ by direct venous inoculation at an independent investigational site. All six developed parasitaemia (GMPPP: 11.4 days, range: 10.4-12.3). Inoculation of PfSPZ was safe. Infection rate and pre-patent period depended on dose, and injection of 3,200 PfSPZ led to a GMPPP similar to CHMI with five PfSPZ-infected mosquitoes. The infectious dose of PfSPZ predicted dosage of radiation-attenuated PfSPZ required for successful vaccination. CONCLUSIONS: IV inoculation of PfSPZ is safe, well tolerated and highly reproducible. It shall further accelerate development of anti-malarial interventions through standardization and facilitation of CHMI. Beyond this, rational dose selection for whole PfSPZ-based immunization and complex study designs are now possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01624961 and NCT01771848

Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis

Immunization of volunteers under chloroquine prophylaxis by bites of *Plasmodium falciparum* sporozoite (PfSPZ)–infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3x monthly immunizations with 7.5 x 10^4 PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 x 10^5 PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI. INTRODUCTIO

Impact of diet on vitamin D status in a Sri Lanka-based sample of pregnant women

Introduction: Vitamin D deficiency is common during pregnancy in Asian countries. However, there is little knowledge about vitamin D status of pregnant mothers and, therefore, supplements are not routinely provided in public clinics and government hospitals in Sri Lanka. Therefore, aim of this study was to assess vitamin D status and adequacy of vitamin D intake in a sample of pregnant mothers. Methods: This was a secondary analysis of existing data from a prospective cohort study. A convenience sample of 89 healthy and non-vitamin D supplemented Sri Lanka-based pregnant mothers was recruited during the third trimester of their pregnancy. Dietary vitamin D intake was assessed through a food frequency questionnaire, while serum was analysed for vitamin D, parathyroid hormone (PTH) and other markers of bone biochemistry. Results: In our sample, average daily dietary intake of vitamin D was 1,289.4 ± 1,225.6 IU/day (range: 56 - 5400 IU). A significant proportion of mothers (45%) consumed < 600 IU of vitamin D per day. More than half of our sample (56.9%) received vitamin D though fortified milk powder and 36% from fish consumption. Most of mothers (69%) consumed small fish and none of them received vitamin D supplementation. There was a significant positive correlation between dietary vitamin D and serum 25-hydroxyvitamin D (25(OH)D) (r = 0.355, P < 0.01). 12.4%, 50.6% and 37% of the mothers were vitamin D deficient, insufficient and sufficient, respectively. We showed a significant difference in levels of dietary vitamin D intake between serum 25(OH)D deficient/insufficient (dietary vitamin D: 1,083.6 ± 1,026.4 IU/day) and 25(OH)D sufficient (dietary vitamin D: 1,638.5 ± 1,456.1 IU/day) groups. Discussion and Conclusion: Dietary intake of vitamin D was inadequate in Sri Lankan non-vitamin D supplemented mothers. Further evaluation of vitamin D status and requirement for supplementation in a nationally representative sample is essential

Successful human infection with P. falciparum using three aseptic Anopheles stephensi mosquitoes: a new model for controlled human malaria infection.

Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18-40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5-333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/µL (range 2-44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8-116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/mosquito (range 3,500-152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.ClinicalTrials.gov NCT00744133 NCT00744133

Laboratory abnormalities recorded during malaria illness after receiving Plasmodium falciparum sporozoites by the bites of 3 aseptic mosquitoes.

<p>AST = aspartate aminotransferase; ALT = alanine aminotransferase; ♂ = male; ♀ = female.</p>*<p>Thrombocytopenia was the only severe laboratory abnormality noted during the study.</p

Maximum intensity of solicited symptoms and signs during days 2–7 after receiving Plasmodium falciparum sporozoites by the bites of 3 aseptic mosquitoes.

<p>Maximum intensity of solicited symptoms and signs during days 2–7 after receiving Plasmodium falciparum sporozoites by the bites of 3 aseptic mosquitoes.</p

Demographic information and study results for participants receiving <i>Plasmodium falciparum</i> sporozoites by the bites of 3 aseptic mosquitoes.

a<p>Previous Study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068969#pone.0068969-Lyke1" target="_blank">[22]</a>.</p>b<p>Current Study.</p

Comparison of geomean quantitative PCR diagnostics following challenge with 3 aseptic mosquitoes versus the traditional 5 mosquito challenge [23].

<p>Error bars represent 95% confidence intervals.</p