No time to die: An in-depth analysis of James Bond's exposure to infectious agents.

Global travelers, whether tourists or secret agents, are exposed to a smörgåsbord of infectious agents. We hypothesized that agents pre-occupied with espionage and counterterrorism may, at their peril, fail to correctly prioritize travel medicine. To examine our hypothesis, we examined adherence to international travel advice during the 86 international journeys that James Bond was observed to undertake in feature films spanning 1962-2021. Scrutinizing these missions involved ∼3113 min of evening hours per author that could easily have been spent on more pressing societal issues. We uncovered above-average sexual activity, often without sufficient time for an exchange of sexual history, with a remarkably high mortality among Bond's sexual partners (27.1; 95% confidence interval 16.4-40.3). Given how inopportune a bout of diarrhea would be in the midst of world-saving action, it is striking that Bond is seen washing his hands on only two occasions, despite numerous exposures to foodborne pathogens. We hypothesize that his foolhardy courage, sometimes purposefully eliciting life-threatening situations, might even be a consequence of Toxoplasmosis. Bond's approach to vector-borne diseases and neglected tropical diseases is erratic, sometimes following travel advice to the letter, but more often dwelling on the side of complete ignorance. Given the limited time Bond receives to prepare for missions, we urgently ask his employer MI6 to take its responsibility seriously. We only live once

Plasmodium falciparum Gametocyte Enrichment in Peripheral Blood Samples by Magnetic Fractionation: Gametocyte Yields and Possibilities to Reuse Columns.

Gametocytes are sexual stage malaria parasites responsible for transmission to mosquitoes. Multiple gametocyte-producing clones may be present in natural infections, but the molecular characterization of gametocytes is challenging. Because of their magnetic properties, gametocyte enrichment can be achieved by magnetic fractionation. This increases detection sensitivity and allows specific genotyping of clones that contribute to malaria transmission. Here, we determined the percentage of Plasmodium falciparum gametocytes successfully bound to magnetic activated cell sorting (MACS) LS columns during magnetic fractionation and assessed whether columns can be reused without risking contamination or affecting column binding efficiency. Bound column fractions were quantified using multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR) for male (pfMGET) and female (CCp4) gametocytes and ring-stage asexual parasites (SBP1). To investigate cross contamination between columns, parasite strain identity was determined by merozoite surface protein 2 genotyping followed by capillary electrophoresis fragment sizing. A reproducible high percentage of gametocytes was bound to MACS LS columns with 94%) of gametocyte enrichment was achieved when columns were used up to five times with lower binding success after eight times (79%). We observed no evidence for cross contamination between columns

Concentration of Plasmodium falciparum gametocytes in whole blood samples by magnetic cell sorting enhances parasite infection rates in mosquito feeding assays.

BACKGROUND: Mosquito-feeding assays are important tools to guide the development and support the evaluation of transmission-blocking interventions. These functional bioassays measure the sporogonic development of gametocytes in blood-fed mosquitoes. Measuring the infectivity of low gametocyte densities has become increasingly important in malaria elimination scenarios. This will pose challenges to the sensitivity and throughput of existing mosquito-feeding assay protocols. Here, different gametocyte concentration methods of blood samples were explored to optimize conditions for detection of positive mosquito infections. METHODS: Mature gametocytes of Plasmodium falciparum were diluted into whole blood samples of malaria-naïve volunteers. Standard centrifugation, Percoll gradient, magnetic cell sorting (MACS) enrichment were compared using starting blood volumes larger than the control (direct) feed. RESULTS: MACS gametocyte enrichment resulted in the highest infection intensity with statistically significant increases in mean oocyst density in 2 of 3 experiments (p = 0.0003; p ≤ 0.0001; p = 0.2348). The Percoll gradient and standard centrifugation procedures resulted in variable infectivity. A significant increase in the proportion of infected mosquitoes and oocyst density was found when larger volumes of gametocyte-infected blood were used with the MACS procedure. CONCLUSIONS: The current study demonstrates that concentration methods of P. falciparum gametocyte-infected whole blood samples can enhance transmission in mosquito-feeding assays. Gametocyte purification by MACS was the most efficient method, allowing the assessment of gametocyte infectivity in low-density gametocyte infections, as can be expected in natural or experimental conditions

Estimating female malaria mosquito age by quantifying Y-linked genes in stored male spermatozoa

Vector control strategies are among the most effective measures to combat mosquito-borne diseases, such as malaria. These strategies work by altering the mosquito age structure through increased mortality of the older female mosquitoes that transmit pathogens. However, methods to monitor changes to mosquito age structure are currently inadequate for programmatic implementation. Female mosquitoes generally mate a single time soon after emergence and draw down spermatozoa reserves with each oviposition cycle. Here, we demonstrate that measuring spermatozoa quantity in female Anopheles mosquitoes is an effective approach to assess mosquito age. Using multiplexed qPCR targeted at male spermatozoa, we show that Y-linked genes in female mosquitoes are exclusively found in the spermatheca, the organ that houses spermatozoa, and the quantity of these gene sequences significantly declines with age. The method can accurately identify mosquitoes more than 10 days old and thus old enough to potentially transmit pathogens harbored in the salivary glands during blood feeding. Furthermore, mosquito populations that differ by 10% in daily survivorship have a high likelihood of being distinguished using modest sample sizes, making this approach scalable for assessing the efficacy of vector intervention control programs

Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR.

BACKGROUND: The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR). METHODS: Cultured Plasmodium falciparum gametocytes were offered to Anopheles stephensi mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured P. falciparum gametocytes. A similar methodology optimized for P. vivax was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with P. vivax. RESULTS: There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured P. falciparum material (agreement 96.9%; kappa = 0.97) and naturally infected P. vivax parasite carriers [agreement 92.4% (kappa = 0.83)]. CONCLUSIONS: The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes

Persistence of mRNA indicative of Plasmodium falciparum ring-stage parasites 42 days after artemisinin and non-artemisinin combination therapy in naturally infected Malians.

BACKGROUND: Malaria control in sub-Saharan Africa relies upon prompt case management with artemisinin-based combination therapy (ACT). Ring-stage parasite mRNA, measured by sbp1 quantitative reverse-transcriptase PCR (qRT-PCR), was previously reported to persist after ACT treatment and hypothesized to reflect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, the persistence of ring-stage parasitaemia following ACT and non-ACT treatment was examined. METHODS: Samples were used from naturally infected Malian gametocyte carriers who received dihydroartemisinin-piperaquine (DP) or sulfadoxine-pyrimethamine (SP-AQ) with or without gametocytocidal drugs. Gametocytes and ring-stage parasites were quantified by qRT-PCR during 42 days of follow-up. RESULTS: At baseline, 89% (64/73) of participants had measurable ring-stage parasite mRNA. Following treatment, the proportion of ring-stage parasite-positive individuals and estimated densities declined for all four treatment groups. Ring-stage parasite prevalence and density was generally lower in arms that received DP compared to SP-AQ. This finding was most apparent days 1, 2, and 42 of follow-up (p < 0.01). Gametocytocidal drugs did not influence ring-stage parasite persistence. Ring-stage parasite density estimates on days 14 and 28 after initiation of treatment were higher among individuals who subsequently experienced recurrent parasitaemia compared to those who remained free of parasites until day 42 after initiation of treatment (pday 14 = 0.011 and pday 28 = 0.068). No association of ring-stage persistence with gametocyte carriage was observed. CONCLUSIONS: The current findings of lower ring-stage persistence after ACT without an effect of gametocytocidal partner drugs affirms the use of sbp1 as ring-stage marker. Lower persistence of ring-stage mRNA after ACT treatment suggests the marker may not reflect dormant parasites whilst it was predictive of re-appearance of parasitaemia

A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance.

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect. Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7_1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes. Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P 0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates. Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness

A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens. Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18–35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays. Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5–12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5–3.9) compared to 5.1 days (95% CI 4.1–6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28). Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development. Funding: Funded by PATH Malaria Vaccine Initiative (MVI). Clinical trial number: NCT02836002

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590