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Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.

Abstract Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Here, the authors show that SARS-CoV-2 infection causes gut microbiome dysbiosis and gut epithelial cell alterations in a mouse model, and correlate dysbiosis observed in COVID-19 patients with blood stream infections, matching reads of bacterial sequences from stool samples to organisms found in the blood.

Abstract Background Arboviruses transmitted by Aedes aegypti including dengue, Zika, and chikungunya are a major global health problem, with over 2.5 billion at risk for dengue alone. There are no licensed antivirals for these infections, and safe and effective vaccines are not yet widely available. Thus, prevention of arbovirus transmission by vector modification is a novel approach being pursued by multiple researchers. However, the field needs high-quality evidence derived from randomized, controlled trials upon which to base the implementation and maintenance of vector control programs. Here, we report the EVITA Dengue trial design (DMID 17-0111), which assesses the efficacy in decreasing arbovirus transmission of an innovative approach developed by the World Mosquito Program for vector modification of Aedes mosquitoes by Wolbachia pipientis. Methods DMID 17-0111 is a cluster-randomized trial in Belo Horizonte, Brazil, with clusters defined by primary school catchment areas. Clusters (n = 58) will be randomized 1:1 to intervention (release of Wolbachia-infected Aedes aegypti mosquitoes) vs. control (no release). Standard vector control activities (i.e., insecticides and education campaigns for reduction of mosquito breeding sites) will continue as per current practice in the municipality. Participants (n = 3480, 60 per cluster) are children aged 6–11 years enrolled in the cluster-defining school and living within the cluster boundaries who will undergo annual serologic surveillance for arboviral infection. The primary objective is to compare sero-incidence of arboviral infection between arms. Discussion DMID 17-0111 aims to determine the efficacy of Wolbachia-infected mosquito releases in reducing human infections by arboviruses transmitted by Aedes aegypti and will complement the mounting evidence for this method from large-scale field releases and ongoing trials. The trial also represents a critical step towards robustness and rigor for how vector control methods are assessed, including the simultaneous measurement and correlation of entomologic and epidemiologic outcomes. Data from this trial will inform further the development of novel vector control methods. Trial registration ClinicalTrials.gov NCT04514107 . Registered on 17 August 2020 Primary sponsor: National Institute of Health, National Institute of Allergy and Infectious Diseases

SARS-CoV-2 infection can lead to progressive pathology in patients with COVID-19, but information for this disease progression is sparse. Here the authors use multi-omics approach to profile the immune responses of patients, assessing immune repertoire and effects of tocilizumab treatments, to find a dyssynchrony between innate and adaptive immunity in progressive COVID-19.

Using a high throughput, random bacterial peptide display approach applied to patient serum samples, Haynes, Kamath, Bozekowski et al identify the antigens and epitopes that elicit a SARS-CoV-2 humoral response. They identify differences depending on disease severity and further in silico analysis suggests decreased epitope signal for Q677P but not for D614G mutant SARSCoV-2 strains.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Limited laboratory capacity in the United States has hindered access to testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has delayed results. To control outbreaks of coronavirus disease [...]

Despite increased diagnostic testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), testing in many countries, including the United States, is still inadequate for slowing the coronavirus disease (COVID-19) [...]

Leptospira was investigated in kidneys (n = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate Leptospira, followed by the LipL32 qPCR to detect Leptospira DNA. The SecY gene region was amplified, sequenced, and analyzed for LipL32 qPCR-positive samples or Leptospira isolates. The overall frequency of isolation of Leptospira spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (p > 0.05). However, with LipL32 qPCR, the overall frequency of Leptospira DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (p = 0.03). Based on 22 SecY sequences, the phylogenetic tree identified the L. interrogans cluster with serovar Icterohaemorrhagiae and the L. borgpetersenii cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of Leptospira spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which L. borgpetersenii serovar Hardjo bovis is not part. Our data show that pathogenic L. interrogans and L. borgpetersenii are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.

Abstract Background COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients. Case presentation We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion. Conclusion Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.

BACKGROUND. The effects of the novel coronavirus disease 2019 (COVID-19) in pregnancy remain relatively unknown. We present a case of second trimester pregnancy with symptomatic COVID-19 complicated by severe preeclampsia and placental abruption. METHODS. We analyzed the placenta for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through molecular and immunohistochemical assays and by and electron microscopy and measured the maternal antibody response in the blood to this infection. RESULTS. SARS-CoV-2 localized predominantly to syncytiotrophoblast cells at the materno-fetal interface of the placenta. Histological examination of the placenta revealed a dense macrophage infiltrate, but no evidence for the vasculopathy typically associated with preeclampsia. CONCLUSION. This case demonstrates SARS-CoV-2 invasion of the placenta, highlighting the potential for severe morbidity among pregnant women with COVID-19. FUNDING. Beatrice Kleinberg Neuwirth Fund and Fast Grant Emergent Ventures funding from the Mercatus Center at George Mason University. The funding bodies did not have roles in the design of the study or data collection, analysis, and interpretation and played no role in writing the manuscript., Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus causing the deadly pandemic of coronavirus disease 2019 (COVID-19). The risks and specific effects of SARS-CoV-2 in pregnant [...]

Summary: Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.

We analyzed feasibility of pooling saliva samples for severe acute respiratory syndrome coronavirus 2 testing and found that sensitivity decreased according to pool size: 5 samples/pool, 7.4% reduction; 10 samples/pool, 11.1%; and 20 samples/pool, 14.8%. When virus prevalence is >2.6%, pools of 5 require fewer tests; when

IntroductionHealthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals. In this study we sought to determine if tracking smell sensitivity and loss using an at-home assessment could identify SARS-CoV-2 infection in HCW.Methods and findingsWe performed a prospective cohort study tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, behavioral at-home assessment of olfaction with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and real-time quantitative polymerase chain reaction testing to identify SARS-CoV-2 infection. Our main measures were the prevalence of smell loss in SARS-CoV-2-positive HCW versus SARS-CoV-2-negative HCW, and timing of smell loss relative to SARS-CoV-2 test positivity. SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. HCW with SARS-CoV-2 infection were more likely to report smell loss than SARS-CoV-2-negative HCW on both the at-home assessment and the screening questionnaire (9/17, 53% vs 105/456, 23%, P < .01). 6/9 (67%) of SARS-CoV-2-positive HCW reporting smell loss reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among SARS-CoV-2-positive HCW who reported smell loss compared to those without smell loss (9/9, 100% vs 3/8, 38%, P < .01).ConclusionsIn this prospective study of HCW, self-reported changes in smell using two different measures were predictive of SARS-CoV-2 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.

Leptospirosis, a zoonosis caused by pathogenic Leptospira, primarily affects tropical, developing regions, especially communities without adequate sanitation. Outbreaks of leptospirosis have been linked with the presence of pathogenic Leptospira in water. In this study, we measured the physicochemical characteristics (temperature, pH, salinity, turbidity, electrical conductivity, and total dissolved solids (TDS)) of surface waters from an urban slum in Salvador, Brazil, and analyzed their associations with the presence and concentration of pathogenic Leptospira reported previously. We built logistic and linear regression models to determine the strength of association between physicochemical parameters and the presence and concentration of Leptospira. We found that salinity, TDS, pH, and type of water were strongly associated with the presence of Leptospira. In contrast, only pH was associated with the concentration of the pathogen in water. The study of physico-chemical markers can contribute to a better understanding of the occurrence of Leptospira in water and to the identification of sources of risk in urban slum environments.

BACKGROUND:Leptospirosis is an important zoonotic disease that causes considerable morbidity and mortality globally, primarily in residents of urban slums. While contact with contaminated water plays a critical role in the transmission of leptospirosis, little is known about the distribution and abundance of pathogenic Leptospira spp. in soil and the potential contribution of this source to human infection. METHODS/PRINCIPAL FINDINGS:We collected soil samples (n = 70) from three sites within an urban slum community endemic for leptospirosis in Salvador, Brazil. Using qPCR of Leptospira genes lipl32 and 16S rRNA, we quantified the pathogenic Leptospira load in each soil sample. lipl32 qPCR detected pathogenic Leptospira in 22 (31%) of 70 samples, though the median concentration among positive samples was low (median = 6 GEq/g; range: 4-4.31×102 GEq/g). We also observed heterogeneity in the distribution of pathogenic Leptospira at the fine spatial scale. However, when using 16S rRNA qPCR, we detected a higher proportion of Leptospira-positive samples (86%) and higher bacterial concentrations (median: 4.16×102 GEq/g; range: 4-2.58×104 GEq/g). Sequencing of the qPCR amplicons and qPCR analysis with all type Leptospira species revealed that the 16S rRNA qPCR detected not only pathogenic Leptospira but also intermediate species, although both methods excluded saprophytic Leptospira. No significant associations were identified between the presence of pathogenic Leptospira DNA and environmental characteristics (vegetation, rat activity, distance to an open sewer or a house, or soil clay content), though samples with higher soil moisture content showed higher prevalences. CONCLUSION/SIGNIFICANCE:This is the first study to successfully quantify the burden of pathogenic Leptospira in soil from an endemic region. Our results support the hypothesis that soil may be an under-recognized environmental reservoir contributing to transmission of pathogenic Leptospira in urban slums. Consequently, the role of soil should be considered when planning interventions aimed to reduce the burden of leptospirosis in these communities.

Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from