Indole-3-pyruvate: Analysis and Control of Tautomerism and Reactivity.

It is well-known in biochemistry that structure confers function, meaning that chemical structural elucidation is critical to truly understanding the function of a given metabolite. Indole-3-pyruvate (IPyA) exists in an equilibrium between the keto and enol tautomeric forms. IPyA is suggested to play a role in immune function; however, determining whether the tautomeric forms function differently can only be studied if an analytical method is capable of distinguishing between the two forms. Herein, we describe the use of UHPLC-HRMS to gain insight into the physical variables that govern IPyA tautomer equilibrium, reactivity, and detection limit. We use hydrogen-deuterium exchange (HDX) to identify enol and keto peaks, and we show that tautomers exhibit a valley of fronting followed by a tailing peak shape (though separation is still attainable) and identical MS/MS spectra. We observed drastically different ratios of keto and enol forms in different solvents, which is an important consideration for in vitro studies. IPyA was found to be highly unstable with accelerated reactivity in peroxides. Through in vitro reactivity studies, IPyA produced a myriad of known and unknown metabolites via nonenzymatic processes, many of which were mapped in vivo via the analysis of human plasma. Finally, we show that vitamin C (ascorbic acid) can slow this reactivity and enable sensitive detection in whole blood.

Syndromic surveillance of population-level COVID-19 burden with cough monitoring in a hospital emergency waiting room.

Syndromic surveillance is an effective tool for enabling the timely detection of infectious disease outbreaks and facilitating the implementation of effective mitigation strategies by public health authorities. While various information sources are currently utilized to collect syndromic signal data for analysis, the aggregated measurement of cough, an important symptom for many illnesses, is not widely employed as a syndromic signal. With recent advancements in ubiquitous sensing technologies, it becomes feasible to continuously measure population-level cough incidence in a contactless, unobtrusive, and automated manner. In this work, we demonstrate the utility of monitoring aggregated cough count as a syndromic indicator to estimate COVID-19 cases. In our study, we deployed a sensor-based platform (Syndromic Logger) in the emergency room of a large hospital. The platform captured syndromic signals from audio, thermal imaging, and radar, while the ground truth data were collected from the hospital's electronic health record. Our analysis revealed a significant correlation between the aggregated cough count and positive COVID-19 cases in the hospital (Pearson correlation of 0.40, p-value < 0.001). Notably, this correlation was higher than that observed with the number of individuals presenting with fever (ρ = 0.22, p = 0.04), a widely used syndromic signal and screening tool for such diseases. Furthermore, we demonstrate how the data obtained from our Syndromic Logger platform could be leveraged to estimate various COVID-19-related statistics using multiple modeling approaches. Aggregated cough counts and other data, such as people density collected from our platform, can be utilized to predict COVID-19 patient visits related metrics in a hospital waiting room, and SHAP and Gini feature importance-based metrics showed cough count as the important feature for these prediction models. Furthermore, we have shown that predictions based on cough counting outperform models based on fever detection (e.g., temperatures over 39°C), which require more intrusive engagement with the population. Our findings highlight that incorporating cough-counting based signals into syndromic surveillance systems can significantly enhance overall resilience against future public health challenges, such as emerging disease outbreaks or pandemics.

CK1 and PP1 regulate Rift Valley fever virus genome replication through L protein phosphorylation.

Rift Valley fever virus (RVFV) is an arbovirus in the Phenuiviridae family identified initially by the large 'abortion storms' observed among ruminants; RVFV can also infect humans. In humans, there is a wide variation of clinical symptoms ranging from subclinical to mild febrile illness to hepatitis, retinitis, delayed-onset encephalitis, or even hemorrhagic fever. The RVFV is a tri-segmented negative-sense RNA virus consisting of S, M, and L segments. The L segment encodes the RNA-dependent RNA polymerase (RdRp), termed the L protein, which is responsible for both viral mRNA synthesis and genome replication. Phosphorylation of viral RdRps is known to regulate viral replication. This study shows that RVFV L protein is serine phosphorylated and identified Casein Kinase 1 alpha (CK1α) and protein phosphatase 1 alpha (PP1α) as L protein binding partners. Inhibition of CK1 and PP1 through small molecule inhibitor treatment, D4476 and 1E7-03, respectively, caused a change in the phosphorylated status of the L protein. Inhibition of PP1α resulted in increased L protein phosphorylation whereas inhibition of CK1α decreased L protein phosphorylation. It was also found that in RVFV infected cells, PP1α localized to the cytoplasmic compartment. Treatment of RVFV infected cells with CK1 inhibitors reduced virus production in both mammalian and mosquito cells. Lastly, inhibition of either CK1 or PP1 reduced viral genomic RNA levels. These data indicate that L protein is phosphorylated and that CK1 and PP1 play a crucial role in regulating the L protein phosphorylation cycle, which is critical to viral RNA production and viral replication.

Evaluating vector competence for Yellow fever in the Caribbean.

The mosquito-borne disease, Yellow fever (YF), has been largely controlled via mass delivery of an effective vaccine and mosquito control interventions. However, there are warning signs that YF is re-emerging in both Sub-Saharan Africa and South America. Imported from Africa in slave ships, YF was responsible for devastating outbreaks in the Caribbean. In Martinique, the last YF outbreak was reported in 1908 and the mosquito Aedes aegypti was incriminated as the main vector. We evaluated the vector competence of fifteen Ae. aegypti populations for five YFV genotypes (Bolivia, Ghana, Nigeria, Sudan, and Uganda). Here we show that mosquito populations from the Caribbean and the Americas were able to transmit the five YFV genotypes, with YFV strains for Uganda and Bolivia having higher transmission success. We also observed that Ae. aegypti populations from Martinique were more susceptible to YFV infection than other populations from neighboring Caribbean islands, as well as North and South America. Our vector competence data suggest that the threat of re-emergence of YF in Martinique and the subsequent spread to Caribbean nations and beyond is plausible.

RTS,S/AS02A Malaria Vaccine-Induced IgG Responses Equally Recognize Native-Like Fucosylated and Nonfucosylated Plasmodium falciparum Circumsporozoite Proteins.

The RTS,S/AS02A malaria vaccine is based on the Plasmodium falciparum circumsporozoite protein (PfCSP), which is O-fucosylated on the sporozoite surface. We determined whether RTS,S/AS02A-induced immunoglobulin G (IgG) antibodies recognize vaccine-like nonfucosylated PfCSP better than native-like fucosylated PfCSP. Similar to previous vaccine trials, RTS,S/AS02A vaccination induced high anti-PfCSP IgG levels associated with malaria protection. IgG recognition of nonfucosylated and fucosylated PfCSP was equivalent, suggesting that PfCSP fucosylation does not affect antibody recognition. Clinical Trials Registration. NCT00197041.

Whole genome sequencing of outbreak strains from 2017 to 2018 reveals an endemic clade of dengue 1 virus in Cameroon.

Dengue fever is expanding as a global public health threat including countries within Africa. For the past few decades, Cameroon has experienced sporadic cases of arboviral infections including dengue fever. Here, we conducted genomic analyses to investigate the origin and phylogenetic profile of Cameroon DENV-1 outbreak strains and predict the impact of emerging therapeutics on these strains. Bayesian and maximum-likelihood phylogenetic inference approaches were employed in virus evolutionary analyses. An in silico analysis was performed to assess the divergence in immunotherapeutic and vaccine targets in the new genomes. Six complete DENV-1 genomes were generated from 50 samples that met a clinical definition for DENV infection. Phylogenetic analyses revealed that the strains from the current study belong to a sub-lineage of DENV-1 genotype V and form a monophyletic taxon with a 2012 strain from Gabon. The most recent common ancestor (TMRCA) of the Cameroon and Gabon strains was estimated to have existed around 2008. Comparing our sequences to the vaccine strains, 19 and 15 amino acid (aa) substitutions were observed in the immuno-protective prM-E protein segments of the Dengvaxia® and TetraVax-DV-TV003 vaccines, respectively. Epitope mapping revealed mismatches in aa residues at positions E155 and E161 located in the epitope of the human anti-DENV-1 monoclonal antibody HMAb 1F4. The new DENV strains constitute a conserved genomic pool of viruses endemic to the Central African region that needs prospective monitoring to track local viral evolution. Further work is needed to ascertain the performance of emerging therapeutics in DENV strains from the African region.

Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism.

Introduction: SARS-CoV-2 subverts host cell processes to facilitate rapid replication and dissemination, and this leads to pathological inflammation. Methods: We used niclosamide (NIC), a poorly soluble anti-helminth drug identified initially for repurposed treatment of COVID-19, which activates the cells' autophagic and lipophagic processes as a chemical probe to determine if it can modulate the host cell's total lipid profile that would otherwise be either amplified or reduced during SARS-CoV-2 infection. Results: Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of SARS-CoV-2 infected Vero E6 cells, especially with triglycerides, which were elevated early during virus replication, but decreased thereafter, as well as plasmalogens, which were elevated at later timepoints during virus replication, but were also elevated under normal cell growth. These findings suggested a complex interplay of lipid profile reorganization involving plasmalogen metabolism. We also observed that NIC treatment of both low and high viral loads does not affect virus entry. Instead, NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which we found elevated during virus infection in the absence of NIC, resulting in a significant reduction in the production of infectious virions. Unexpectedly, at higher viral loads, NIC treatment also resulted in elevated triglyceride levels, and induced significant changes in phospholipid metabolism. Discussion: We posit that future screens of approved or new partner drugs should prioritize compounds that effectively counter SARS-CoV-2 subversion of lipid metabolism, thereby reducing virus replication, egress, and the subsequent regulation of key lipid mediators of pathological inflammation.

Impact of malaria diagnostic choice on monitoring of Plasmodium falciparum prevalence estimates in the Democratic Republic of the Congo and relevance to control programs in high-burden countries.

Malaria programs rely upon a variety of diagnostic assays, including rapid diagnostic tests (RDTs), microscopy, polymerase chain reaction (PCR), and bead-based immunoassays (BBA), to monitor malaria prevalence and support control and elimination efforts. Data comparing these assays are limited, especially from high-burden countries like the Democratic Republic of the Congo (DRC). Using cross-sectional and routine data, we compared diagnostic performance and Plasmodium falciparum prevalence estimates across health areas of varying transmission intensity to illustrate the relevance of assay performance to malaria control programs. Data and samples were collected between March-June 2018 during a cross-sectional household survey across three health areas with low, moderate, and high transmission intensities within Kinshasa Province, DRC. Samples from 1,431 participants were evaluated using RDT, microscopy, PCR, and BBA. P. falciparum parasite prevalence varied between diagnostic methods across all health areas, with the highest prevalence estimates observed in Bu (57.4-72.4% across assays), followed by Kimpoko (32.6-53.2%), and Voix du Peuple (3.1-8.4%). Using latent class analysis to compare these diagnostic methods against an "alloyed gold standard," the most sensitive diagnostic method was BBA in Bu (high prevalence) and Voix du Peuple (low prevalence), while PCR diagnosis was most sensitive in Kimpoko (moderate prevalence). RDTs were consistently the most specific diagnostic method in all health areas. Among 9.0 million people residing in Kinshasa Province in 2018, the estimated P. falciparum prevalence by microscopy, PCR, and BBA were nearly double that of RDT. Comparison of malaria RDT, microscopy, PCR, and BBA results confirmed differences in sensitivity and specificity that varied by endemicity, with PCR and BBA performing best for detecting any P. falciparum infection. Prevalence estimates varied widely depending on assay type for parasite detection. Inherent differences in assay performance should be carefully considered when using community survey and surveillance data to guide policy decisions.

Passive Monitoring of Crowd-Level Cough Counts in Waiting Areas produces Reliable Syndromic Indicator for Total COVID-19 Burden in a Hospital Emergency Clinic.

Syndromic surveillance is an effective tool for enabling the timely detection of infectious disease outbreaks and facilitating the implementation of effective mitigation strategies by public health authorities. While various information sources are currently utilized to collect syndromic signal data for analysis, the aggregated measurement of cough, an important symptom for many illnesses, is not widely employed as a syndromic signal. With recent advancements in ubiquitous sensing technologies, it becomes feasible to continuously measure population-level cough incidence in a contactless, unobtrusive, and automated manner. In this work, we demonstrate the utility of monitoring aggregated cough count as a syndromic indicator to estimate COVID-19 cases. In our study, we deployed a sensor-based platform (Syndromic Logger) in the emergency room of a large hospital. The platform captured syndromic signals from audio, thermal imaging, and radar, while the ground truth data were collected from the hospital's electronic health record. Our analysis revealed a significant correlation between the aggregated cough count and positive COVID-19 cases in the hospital (Pearson correlation of 0.40, p-value < 0.001). Notably, this correlation was higher than that observed with the number of individuals presenting with fever (ρ = 0.22, p = 0.04), a widely used syndromic signal and screening tool for such diseases. Furthermore, we demonstrate how the data obtained from our Syndromic Logger platform could be leveraged to estimate various COVID-19-related statistics using multiple modeling approaches. Our findings highlight the efficacy of aggregated cough count as a valuable syndromic indicator associated with the occurrence of COVID-19 cases. Incorporating this signal into syndromic surveillance systems for such diseases can significantly enhance overall resilience against future public health challenges, such as emerging disease outbreaks or pandemics.

A mixed methods study assessing the adoption potential of a saliva-based malaria rapid test in the Democratic Republic of Congo.

BACKGROUND: The reliance on blood for thin and thick blood smear microscopy-using a relatively invasive procedure has presented challenges to the use of reliable diagnostic tests in non-clinical settings at the point-of-need (PON). To improve the capacity of non-blood-based rapid diagnostic tests to confirm subclinical infections, and thereby identify and quantify the human reservoir at the PON, a cross-sectoral collaboration between university researchers and commercial partners produced an innovative, non-invasive saliva-based RDT capable of identifying novel, non-hrp2/3 parasite biomarkers. While this new saliva-based malaria asymptomatic and asexual rapid test (SMAART-1) shows increased detection sensitivity and precision potential by identifying a new P. falciparum protein marker (PSSP17), appraising its utility in the field-particularly with respect to its adoption potential with children and adults in high risk, endemic regions-is necessary to warrant its continued development. METHODS: The purpose of this study was to assess the acceptability and adoption potential of the SMAART-1 at select PON sites in the Kinshasa Province. Teachers, community health workers, nurses, and laboratory technicians participated in data collection at three distinct community sites in Kinshasa Province, Democratic Republic of the Congo. Three data collection methods were utilized in this mixed methods study to provide an overarching acceptability evaluation of the SMAART-1 at PON field sites: observation checklists of SMAART-1 implementation, focus group discussions, and surveys with local health care practitioners-particularly teachers and community health workers. RESULTS: Findings indicate participants were interested in and supportive of the SMAART-1 protocol, with approximately 99% of the participants surveyed indicating that they either "agreed" or "strongly agreed" with the statement that they "would use the saliva-based malaria asymptomatic rapid test as part of a community malaria detection and treatment programme." Data also suggest that the protocol was broadly appealing for its testing sensitivity and ease of use. CONCLUSIONS: The SMAART-1 protocol's clinically reliable results demonstrate a promising new level of sensitivity and precision for detecting parasite biomarkers. This study's mixed-methods assessment of the protocol's utility and adoption potential in the field, with a target user audience, advances its development and points to opportunities to formalize and expand evaluation efforts.

Plasmodium falciparum with pfhrp2/3 Deletion Not Detected in a 2018-2021 Malaria Longitudinal Cohort Study in Kinshasa Province, Democratic Republic of the Congo.

Histidine-rich protein 2- (HRP2-) based rapid diagnostic tests (RDTs) are widely used to detect Plasmodium falciparum in sub-Saharan Africa. Reports of parasites with pfhrp2 and/or pfhrp3 (pfhrp2/3) gene deletions in Africa raise concerns about the long-term viability of HRP2-based RDTs. We evaluated changes in pfhrp2/3 deletion prevalence over time using a 2018-2021 longitudinal study of 1,635 enrolled individuals in Kinshasa Province, Democratic Republic of the Congo (DRC). Samples collected during biannual household visits with ≥ 100 parasites/µL by quantitative real-time polymerase chain reaction were genotyped using a multiplex real-time PCR assay. Among 2,726 P. falciparum PCR-positive samples collected from 993 participants during the study period, 1,267 (46.5%) were genotyped. No pfhrp2/3 deletions or mixed pfhrp2/3-intact and -deleted infections were identified in our study. Pfhrp2/3-deleted parasites were not detected in Kinshasa Province; ongoing use of HRP2-based RDTs is appropriate.

Adipokinetic hormone signaling in the malaria vector Anopheles gambiae facilitates Plasmodium falciparum sporogony.

An obligatory step in the complex life cycle of the malaria parasite is sporogony, which occurs during the oocyst stage in adult female Anopheles mosquitoes. Sporogony is metabolically demanding, and successful oocyst maturation is dependent on host lipids. In insects, lipid energy reserves are mobilized by adipokinetic hormones (AKHs). We hypothesized that Plasmodium falciparum infection activates Anopheles gambiae AKH signaling and lipid mobilization. We profiled the expression patterns of AKH pathway genes and AgAkh1 peptide levels in An. gambiae during starvation, after blood feeding, and following infection and observed a significant time-dependent up-regulation of AKH pathway genes and peptide levels during infection. Depletion of AgAkh1 and AgAkhR by RNAi reduced salivary gland sporozoite production, while synthetic AgAkh1 peptide supplementation rescued sporozoite numbers. Inoculation of uninfected female mosquitoes with supernatant from P. falciparum-infected midguts activated AKH signaling. Clearly, identifying the parasite molecules mediating AKH signaling in P. falciparum sporogony is paramount.

Needs Assessment of Southeastern United States Vector Control Agencies: Capacity Improvement Is Greatly Needed to Prevent the Next Vector-Borne Disease Outbreak.

A national 2017 vector control capacity survey was conducted to assess the United States' (U.S.'s) ability to prevent emerging vector-borne disease. Since that survey, the southeastern U.S. has experienced continued autochthonous exotic vector-borne disease transmission and establishment of invasive vector species. To understand the current gaps in control programs and establish a baseline to evaluate future vector control efforts for this vulnerable region, a focused needs assessment survey was conducted in early 2020. The southeastern U.S. region was targeted, as this region has a high probability of novel vector-borne disease introduction. Paper copies delivered in handwritten envelopes and electronic copies of the survey were delivered to 386 unique contacts, and 150 returned surveys were received, corresponding to a 39% response rate. Overall, the survey found vector control programs serving areas with over 100,000 residents and those affiliated with public health departments had more core capabilities compared to smaller programs and those not affiliated with public health departments. Furthermore, the majority of vector control programs in this region do not routinely monitor for pesticide resistance. Taken as a whole, these results suggest that the majority of the southeastern U.S. is vulnerable to vector-borne disease outbreaks. Results from this survey raise attention to the critical need of providing increased resources to bring all vector control programs to a competent level, ensuring that public health is protected from the threat of vector-borne disease.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.

BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).

Intrinsic variation in the vertically transmitted core virome of the mosquito Aedes aegypti.

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level.

Seasonality and transmissibility of Plasmodium ovale in Bagamoyo District, Tanzania.

BACKGROUND: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed. METHODS: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development. RESULTS: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae. CONCLUSIONS: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum.

Imported Dengue Case Numbers and Local Climatic Patterns Are Associated with Dengue Virus Transmission in Florida, USA.

Aedes aegypti mosquitoes are the main vector of dengue viruses globally and are present throughout much of the state of Florida (FL) in the United States of America. However, local transmission of dengue viruses in FL has mainly occurred in the southernmost counties; specifically Monroe and Miami-Dade counties. To get a better understanding of the ecologic risk factors for dengue fever incidence throughout FL, we collected and analyzed numerous environmental factors that have previously been connected to local dengue cases in disease-endemic regions. We analyzed these factors for each county-year in FL, between 2009-2019, using negative binomial regression. Monthly minimum temperature of 17.5-20.8 °C, an average temperature of 26.1-26.7 °C, a maximum temperature of 33.6-34.7 °C, rainfall between 11.4-12.7 cm, and increasing numbers of imported dengue cases were associated with the highest risk of dengue incidence per county-year. To our knowledge, we have developed the first predictive model for dengue fever incidence in FL counties and our findings provide critical information about weather conditions that could increase the risk for dengue outbreaks as well as the important contribution of imported dengue cases to local establishment of the virus in Ae. aegypti populations.

A molecular surveillance-guided vector control response to concurrent dengue and West Nile virus outbreaks in a COVID-19 hotspot of Florida.

Background: Simultaneous dengue virus (DENV) and West Nile virus (WNV) outbreaks in Florida, USA, in 2020 resulted in 71 dengue virus serotype 1 and 86 WNV human cases. We hypothesized that we would find a number of DENV-1 positive mosquito pools, and that the distribution of these arbovirus-positive mosquito pools would be associated with those neighborhoods for which imported DENV cases have been recently reported in 2019 and 2020. Methods: We collected and screened Aedes aegypti, Ae. albopictus, Anopheles crucians, Culex coronator, Cx. nigripalpus, and Cx. quinquefasciatus mosquitoes from Miami-Dade County (Florida) for DENV and WNV by rRT-qPCR. Spatial statistical analyses were performed to capture positive mosquito pool distribution in relation to land use, human demography, environmental variables, mosquito trap placement and reported human travel associated DENV cases to guide future mosquito control outbreak responses. Findings: A rapid screen of 7,668 mosquitoes detected four DENV serotype 2 (DENV-2), nine DENV-4 and nine WNV-positive mosquito pools, which enabled swift and targeted abatement of trap sites by mosquito control. As expected, DENV-positive pools were in urban areas; however, we found WNV-positive mosquito pools in agricultural and recreational areas with no historical reports of WNV transmission. Interpretation: These findings demonstrate the importance of proactive arbovirus surveillance in mosquito populations to prevent and control outbreaks, particularly when other illnesses (e.g., COVID-19), which present with similar symptoms, are circulating concurrently. Growing evidence for substantial infection prevalence of dengue in mosquitoes in the absence of local index cases suggests a higher level of dengue endemicity in Florida than previously thought. Funding: This research was supported in part by U.S. Centers for Disease Control and Prevention (CDC) grant 1U01CK000510-03, Southeastern Regional Center of Excellence in Vector Borne Diseases Gateway Program.

Geographic Partitioning of Dengue Virus Transmission Risk in Florida.

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern.

Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has the potential to transform diagnostics due to its programmability. However, many of the CRISPR-based detection methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. A complete one-pot detection reaction using alternative Cas effector endonucleases has been proposed to overcome these challenges. Yet, current approaches using Alicyclobacillus acidiphilus Cas12b (AapCas12b) are limited by its thermal instability at optimum reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction temperatures. Herein, we demonstrate that a novel Cas12b from Brevibacillus sp. SYP-B805 (referred to as BrCas12b) has robust trans-cleavage activity at ideal RT-LAMP conditions. A competitive profiling study of BrCas12b against Cas12b homologs from other bacteria genera underscores the potential of BrCas12b in the development of new diagnostics. As a proof-of-concept, we incorporated BrCas12b into an RT-LAMP-mediated one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs) to enable rapid, differential detection of SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) in 205 clinical samples. Notably, a BrCas12b detection signal was observed within 1-3 minutes of amplification, achieving an overall 98.1% specificity, 91.2% accuracy, and 88.1% sensitivity within 30 minutes. Significantly, for samples with high viral load (C t value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, we combined the lyophilized one-pot reagents with a portable multiplexing device capable of interpreting fluorescence signals at a fraction of the cost of a qPCR system. With relaxed design requirements, one-pot detection, and simple instrumentation, this assay has the capability to advance future diagnostics.