Assessing the daily natural history of asymptomatic Plasmodium infections in adults and older children in Katakwi, Uganda: a longitudinal cohort study.

BACKGROUND: Low-density asymptomatic Plasmodium infections are prevalent in endemic areas, but little is known about their natural history. The trajectories of these infections and their propensity to fluctuate to undetectable densities can affect detection in clinical trials and field studies. We aimed to classify the natural history of these infections in a high transmission area over 29 days. METHODS: In this longitudinal cohort study, we enrolled healthy, malaria-asymptomatic, afebrile, adults (age 18-59 years) and older children (age 8-17 years) in Katakwi District, Uganda, who were negative for Plasmodium infection on rapid diagnostic tests. Participants were instructed to self-collect one dried blood spot (DBS) per day for a maximum of 29 days. We excluded people if they were pregnant or taking antimalarials. During weekly clinic visits, staff collected a DBS and a 4 mL sample of venous blood. We analysed DBSs by Plasmodium 18S rRNA quantitative RT-PCR (qRT-PCR). We classified DBS by infection type as negative, P falciparum, non-P falciparum, or mixed. We plotted infection type over time for each participant and categorised trajectories as negative, new, cleared, chronic, or indeterminate infections. To estimate the effect of single timepoint sampling, we calculated the daily prevalence for each study day and estimated the number of infections that would have been detected in our population if sampling frequency was reduced. FINDINGS: Between April 9 and May 20, 2021, 3577 DBSs were collected by 128 (40 male adults, 60 female adults, 12 male children, and 16 female children) study participants. 2287 (64%) DBSs were categorised as negative, 751 (21%) as positive for P falciparum, 507 (14%) as positive for non-P falciparum, and 32 (1%) as mixed infections. Daily Plasmodium prevalence in the population ranged from 45·3% (95% CI 36·6-54·1) at baseline to 30·3% (21·9-38·6) on day 24. 37 (95%) of 39 P falciparum and 35 (85%) of 41 non-P falciparum infections would have been detected with every other day sampling, whereas, with weekly sampling, 35 (90%) P falciparum infections and 31 (76%) non-P falciparum infections would have been detected. INTERPRETATION: Parasite dynamics and species are highly variable among low-density asymptomatic Plasmodium infections. Sampling every other day or every 3 days detected a similar proportion of infections as daily sampling, whereas testing once per week or even less frequently could misclassify up to a third of the infections. Even using highly sensitive diagnostics, single timepoint testing might misclassify the true infection status of an individual. FUNDING: US National Institutes of Health and Bill and Melinda Gates Foundation.

Plasmodium knowlesi in pig-tailed macaques: a potential new model for malaria vaccine research.

BACKGROUND: Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites. However, rhesus are not natural hosts for P. knowlesi, and there is interest in identifying alternative hosts for vaccine research. The study team previously reported that pig-tailed macaques (PTM), a natural host for P. knowlesi, could be challenged with cryopreserved P. knowlesi sporozoites (PkSPZ), with time to blood stage infection equivalent to in rhesus. Here, additional exploratory studies were performed to evaluate PTM as potential hosts for malaria vaccine studies. The aim was to further characterize the parasitological and veterinary health outcomes after PkSPZ challenge in this macaque species. METHODS: Malaria-naïve PTM were intravenously challenged with 2.5 × 103 PkSPZ and monitored for blood stage infection by Plasmodium 18S rRNA RT-PCR and thin blood smears. Disease signs were evaluated by daily observations, complete blood counts, serum chemistry tests, and veterinary examinations. After anti-malarial drug treatment, a subset of animals was re-challenged and monitored as above. Whole blood gene expression analysis was performed on selected animals to assess host response to infection. RESULTS: In naïve animals, the kinetics of P. knowlesi blood stage replication was reproducible, with parasite burden rising linearly during an initial acute phase of infection from 6 to 11 days post-challenge, before plateauing and transitioning into a chronic low-grade infection. After re-challenge, infections were again reproducible, but with lower blood stage parasite densities. Clinical signs of disease were absent or mild and anti-malarial treatment was not needed until the pre-defined study day. Whole blood gene expression analysis identified immunological changes associated with acute and chronic phases of infection, and further differences between initial challenge versus re-challenge. CONCLUSIONS: The ability to challenge PTM with PkSPZ and achieve reliable blood stage infections indicate this model has significant potential for malaria vaccine studies. Blood stage P. knowlesi infection in PTM is characterized by low parasite burdens and a benign disease course, in contrast with the virulent P. knowlesi disease course commonly reported in rhesus macaques. These findings identify new opportunities for malaria vaccine research using this natural host-parasite combination.

Plasmodium 18S Ribosomal RNA Biomarker Clearance After Food and Drug Administration-Approved Antimalarial Treatment in Controlled Human Malaria Infection Trials.

Background: Sensitive molecular assays, such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of Plasmodium 18S ribosomal RNA (rRNA), are increasingly the primary method of detecting infections in controlled human malaria infection (CHMI) trials. However, thick blood smears (TBSs) remain the main method for confirming clearance of parasites after curative treatment, in part owing to uncertainty regarding biomarker clearance rates. Methods: For this analysis, 18S rRNA qRT-PCR data were compiled from 127 Plasmodium falciparum-infected participants treated with chloroquine or atovaquone-proguanil in 6 CHMI studies conducted in Seattle, Washington, over the past decade. A survival analysis approach was used to compare biomarker and TBS clearance times among studies. The effect of the parasite density at which treatment was initiated on clearance time was estimated using linear regression. Results: The median time to biomarker clearance was 3 days (interquartile range, 3-5 days), while the median time to TBS clearance was 1 day (1-2 days). Time to biomarker clearance increased with the parasite density at which treatment was initiated. Parasite density did not have a significant effect on TBS clearance. Conclusions: The Plasmodium 18S rRNA biomarker clears quickly and can be relied on to confirm the adequacy of Food and Drug Administration-approved treatments in CHMI studies at nonendemic sites.

Rethinking detection of pre-existing and intervening Plasmodium infections in malaria clinical trials.

Pre-existing and intervening low-density Plasmodium infections complicate the conduct of malaria clinical trials. These infections confound infection detection endpoints, and their immunological effects may detract from intended vaccine-induced immune responses. Historically, these infections were often unrecognized since infrequent and often analytically insensitive parasitological testing was performed before and during trials. Molecular diagnostics now permits their detection, but investigators must weigh the cost, complexity, and personnel demands on the study and the laboratory when scheduling such tests. This paper discusses the effect of pre-existing and intervening, low-density Plasmodium infections on malaria vaccine trial endpoints and the current methods employed for their infection detection. We review detection techniques, that until recently, provided a dearth of cost-effective strategies for detecting low density infections. A recently deployed, field-tested, simple, and cost-effective molecular diagnostic strategy for detecting pre-existing and intervening Plasmodium infections from dried blood spots (DBS) in malaria-endemic settings is discussed to inform new clinical trial designs. Strategies that combine sensitive molecular diagnostic techniques with convenient DBS collections and cost-effective pooling strategies may enable more thorough and informative infection monitoring in upcoming malaria clinical trials and epidemiological studies.

Feasibility of community at-home dried blood spot collection combined with pooled reverse transcription PCR as a viable and convenient method for malaria epidemiology studies.

BACKGROUND: Many Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia. Self-collected dried blood spots (DBS) analysed using pooled and individual quantitative reverse transcription polymerase chain reaction (qRT-PCR) provide such a solution. Here, the feasibility and acceptability of daily at-home DBS collections for qRT-PCR was studied to better understand low-density infections. METHODS: Rapid diagnostic test (RDT)-negative individuals in Katakwi District, northeastern Uganda, were recruited between April and May 2021. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits. Participants were trained in DBS collection and asked to collect six DBS weekly between clinic visits. Opinions about the collection process were solicited using daily Diary Cards and a Likert scale survey at the final study visit. Venous blood and DBS were analysed by Plasmodium 18S rRNA qRT-PCR. The number of participants completing the study, total DBS collected, and opinions of the process were analysed to determine compliance and acceptability. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for at-home vs. clinic-collected DBS and venous blood to assess quality and accuracy of at-home collected samples. RESULTS: One-hundred two adults and 29 children were enrolled, and 95 and 26 completed the study, respectively. Three individuals withdrew due to pain or inconvenience of procedures. Overall, 96% of participants collected ≥ 16 of 24 at-home DBS, and 87% of DBS contained ≥ 40 µL of blood. The procedure was well tolerated and viewed favourably by participants. At-home collected DBS were acceptable for qRT-PCR and showed less than a one qRT-PCR cycle threshold shift in the human control mRNA compared to clinic-collected DBS. Correlation between Plasmodium falciparum 18S rRNA from paired whole blood and DBS was high (R = 0.93). CONCLUSIONS: At-home DBS collection is a feasible, acceptable, and robust method to obtain blood to evaluate the natural history of low-density Plasmodium infections by qRT-PCR.

Distributions of Invasive Arthropods across Heterogeneous Urban Landscapes in Southern California: Aridity as a Key Component of Ecological Resistance.

Urban systems often support large numbers of non-native species, but due to the heterogeneity of urban landscapes, species are not evenly distributed. Understanding the drivers of ecological resistance in urban landscapes may help to identify habitats that are most resistant to invasion, and inform efforts to model and conserve native biodiversity. We used pitfall traps to survey non-native ground-dwelling arthropods in three adjacent, low-elevation habitat types in southern California: California sage scrub, non-native grassland, and suburban development. We found that non-native species were fewer and less widely distributed in the sage scrub and grassland habitats. Due to the proximity of our sites, differences in propagule pressure is an unlikely explanation. Instead, we suggest that the absence of water subsidies in the sage scrub and grassland habitats increases those habitats' resistance to arthropod invasions. Comparisons to studies conducted at fragments closer to the coast provide further support for the relationship between aridity and invasibility in southern California. Our findings highlight that inland fragments are important for conserving native arthropod diversity, that models of non-native species distributions in arid and semi-arid urban systems should include aridity measures, and that reducing resource subsidies across the region is critical to mitigating spread of non-natives.