Development and validation of a rapid and easy-to-perform point-of-care lateral flow immunoassay (LFIA) for the detection of SARS-CoV-2 spike protein.

Development and validation of rapid and easy-to-perform diagnostics continue to be a high priority during the current COVID-19 pandemic. Although vaccines are now widely available, early detection and consistent transmission control provide ideal means to mitigate the spread of SARS-CoV-2. Nucleic acid-based real-time PCR tests are widely acknowledged as the gold standard for reliable diagnosis of COVID-19 infection. These tests are based on detecting viable or nonviable viral nucleic acids. SARS-CoV-2 spike protein is an alternative and ideal target for SARS-CoV-2 diagnosis in the early phase of infection, but point-of-care kits to detect the SARS-CoV-2 spike protein are limited. Here we describe a rapid and convenient method based on Lateral Flow Immunoassay (LFIA) to detect SARS-CoV-2 spike proteins, including SARS-CoV-2 variants (A.23.1, B.1.1.1, 1.617.2, B.1.1.7, B.1.351, P.1, N501Y, R.1, P681H, P3, UK, and South African) within 5 to 10 minutes. We generated highly specific monoclonal antibodies (mAbs) against rationally designed SARS-CoV-2 spike protein. Matched pair mAbs were selected by epitope mapping and employed as antigen capture reagents by spotting onto a nitrocellulose membrane and as detector reagents by conjugation with colloidal gold nanoparticles. We evaluated the performance of the LFIA using recombinant spike proteins of SARS-CoV-2 and several SARS-CoV-2 variants. The specificity of the LFIA was assessed using heat-inactivated SARS-CoV-2 and related human coronaviruses (HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63) and an FDA-approved respiratory pathogens (RP) panel. The assay exhibited 98% specificity and acceptable performance with respect to the minimum limit of detection (25 ng/test) in validation tests. This new LFIA provides improved performance for the early diagnosis of SARS-CoV-2, particularly for home monitoring and in situations with limited access to molecular methods.

Dynamics of parasite growth in genetically diverse Plasmodium falciparum isolates.

Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical malaria isolate, resulting in complex growth interactions and variations in phenotype. To elucidate the dynamics of parasite growth in multiclonal isolates, we measured growth rates (GRs) of three Plasmodium falciparum Cambodian isolates, including IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285), and parasite lineages previously cloned from each of these isolates by limiting dilution. Following synchronization, in vitro cultures of each parasite line were maintained over four consecutive asexual cycles (192 h), with thin smears prepared at each 48-h cycle to estimate GR and fold change in parasitemia (FCP). Cell cycle time (CCT), the duration it takes for ring-stage parasites to develop into mature schizonts, was measured by monitoring the development of 0-3-h post-invasion rings for up to 52 h post-incubation. Laboratory lines 3D7 (MRA-102) and Dd2 (MRA-150) were used as controls. Significant differences in GR, FCP, and CCT were observed between parasite isolates and clonal lineages from each isolate. The parasite lines studied here have well-defined growth phenotypes and will facilitate basic malaria research and development of novel malaria interventions. These lines are available to malaria researchers through the MR4 collection of NIAID's BEI Resources Program.

A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium.

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Proteomic analysis reveals pathogen-derived biomarkers of acute babesiosis in erythrocytes, plasma, and urine of infected hamsters.

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.

Update on BEI Resources for Parasitology and Arthropod Vector Research.

BEI Resources has contributed to the advancement of parasitic diseases research for over 16 years. The accessibility of our reference strains and reagents is relevant to the development of new therapeutics and vaccines. Here we provide a resource update with emphasis on the new assets for toxoplasmosis and vector research.

Analysis of the Babesia microti proteome in infected red blood cells by a combination of nanotechnology and mass spectrometry.

Proteomics of Babesia microti has lagged behind other apicomplexans despite recent genome and transcriptome studies. Here, we used a combination of nanotechnology and mass spectrometry to provide a proteomic profile of B. microti acute infection. We identified ∼500 parasite proteins in blood with functions such as transport, carbohydrate and energy metabolism, proteolysis, DNA and RNA metabolism, signaling, translation, lipid biosynthesis, and motility and invasion. We also identified surface antigens with roles in the immune response to the parasite. This first evaluation of the B. microti proteome in erythrocytes provides information for the study of intracellular survival and development of diagnostic tools using mass spectrometry.

Establishment of a continuous in vitro culture of Babesia duncani in human erythrocytes reveals unusually high tolerance to recommended therapies.

Human babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia Clinical cases caused by Babesia duncani have been associated with high parasite burden, severe pathology, and death. In both mice and hamsters, the parasite causes uncontrolled fulminant infections, which ultimately lead to death. Resolving these infections requires knowledge of B. duncani biology, virulence, and susceptibility to anti-infectives, but little is known and further research is hindered by a lack of relevant model systems. Here, we report the first continuous in vitro culture of B. duncani in human red blood cells. We show that during its asexual cycle within human erythrocytes, B. duncani develops and divides to form four daughter parasites with parasitemia doubling every ∼22 h. Using this in vitro culture assay, we found that B. duncani has low susceptibility to the four drugs recommended for treatment of human babesiosis, atovaquone, azithromycin, clindamycin, and quinine, with IC50 values ranging between 500 nm and 20 µm These data suggest that current practices are of limited effect in treating the disease. We anticipate this new disease model will set the stage for a better understanding of the biology of this parasite and will help guide better therapeutic strategies to treat B. duncani-associated babesiosis.

Phylogenomics of 'Discosea': A new molecular phylogenetic perspective on Amoebozoa with flat body forms.

The majority of amoeboid lineages with flattened body forms are placed under a taxonomic hypothetical class 'Discosea' sensu Smirnov et al. (2011), which encompasses some of the most diverse morphs within Amoebozoa. However, its taxonomy and phylogeny is poorly understood. This is partly due to lack of support in studies that are based on limited gene sampling. In this study we use a phylogenomic approach including newly-generated RNA-Seq data and comprehensive taxon sampling to resolve the phylogeny of 'Discosea'. Our analysis included representatives from all orders of 'Discosea' and up to 550 genes, the largest gene sampling in Amoebozoa to date. We conducted extensive analyses to assess the robustness of our resulting phylogenies to effects of missing data and outgroup choice using probabilistic methods. All of our analyses, which explore the impact of varying amounts of missing data, consistently recover well-resolved and supported groups of Amoebozoa. Our results neither support the monophyly nor dichotomy of 'Discosea' as defined by Smirnov et al. (2011). Rather, we recover a robust well-resolved clade referred to as Eudiscosea encompassing the majority of discosean orders (seven of the nine studied here), while the Dactylopodida, Thecamoebida and Himatismenida, previously included in 'Discosea,' are non-monophyletic. We also recover novel relationships within the Eudiscosea that are largely congruent with morphology. Our analyses enabled us to place some incertae sedis lineages and previously unstable lineages such as Vermistella, Mayorella, Gocevia, and Stereomyxa. We recommend some phylogeny-based taxonomic amendments highlighting the new findings of this study and discuss the evolution of the group based on our current understanding.

Sapocribrum chincoteaguense n. gen. n. sp.: A Small, Scale-bearing Amoebozoan with Flabellinid Affinities.

The isolate American Type Culture Collection (ATCC)® 50979™ is a small amoebozoan whose actin gene was previously characterized, but did not allow a stable phylogenetic placement. This isolate was originally mis-identified upon deposition, and subsequently mis-illustrated in a recent publication. Here, we provide both a detailed morphological description as well as additional molecular analyses in order to clarify the isolate's phylogenetic relationships. The amoeba is minute (less than 5 µm), and presents the behavior of staying in a fixed location, while emitting one or two thin pseudopods. Transmission electron microscopy reveals that the cell is covered in a layer with embedded scales, giving the cell an armored appearance. Molecular phylogenetic analyses of data (actin, alpha- and beta-tubulin, elongation factor 2, and 14-3-3) from transcriptomes of this and four other isolates reveals that ATCC® 50979(™) is closely related to the recently described Squamamoeba japonica and in a novel, stable clade. Due to the unique nature of the scale covering, as well as other gross morphological characters and the molecular phylogenetic analyses, we formally describe the isolate as Sapocribrum chincoteaguense n. gen. n. sp.

Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani.

Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.

The others: our biased perspective of eukaryotic genomes.

Understanding the origin and evolution of the eukaryotic cell and the full diversity of eukaryotes is relevant to many biological disciplines. However, our current understanding of eukaryotic genomes is extremely biased, leading to a skewed view of eukaryotic biology. We argue that a phylogeny-driven initiative to cover the full eukaryotic diversity is needed to overcome this bias. We encourage the community: (i) to sequence a representative of the neglected groups available at public culture collections, (ii) to increase our culturing efforts, and (iii) to embrace single cell genomics to access organisms refractory to propagation in culture. We hope that the community will welcome this proposal, explore the approaches suggested, and join efforts to sequence the full diversity of eukaryotes.

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae).

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.

Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes.

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).

Evidence for a previously unrecognized mycobacterial endosymbiont in Acanthamoeba castellanii strain Ma (ATCC ® 50370 ™).

We describe the isolation of a mycobacterium from Acanthamoeba castellanii strain Ma (ATCC(®) 50370(™)). The mycobacterium resides within vacuoles of A. castellanii, can be cultured by routine methodologies, and is a member of the Mycobacterium avium complex. Previously unrecognized mycobacterial endosymbionts are likely common among strains of Acanthamoeba housed at culture collections.

Infection with Toxoplasma gondii results in dysregulation of the host cell cycle.

Mammalian cells infected with Toxoplasma gondii are characterized by a profound reprogramming of gene expression. We examined whether such transcriptional responses were linked to changes in the cell cycle of the host. Human foreskin fibroblasts (HFFs) in the G(0)/G(1) phase of the cell cycle were infected with T. gondii and FACS analysis of DNA content was performed. Cell cycle profiles revealed a promotion into the S phase followed by an arrest towards the G(2)/M boundary with infection. This response was markedly different from that of growth factor stimulation which caused cell cycle entry and completion. Transcriptional profiles of T. gondii-infected HFF showed sustained increases in transcripts associated with a G(1)/S transition and DNA synthesis coupled to an abrogation of cell cycle regulators critical in G(2)/M transition relative to growth factor stimulation. These divergent responses correlated with a distinct temporal modulation of the critical cell cycle regulator kinase ERK by infection. While the kinetics of ERK phosphorylation by EGF showed rapid and sustained activation, infected cells displayed an oscillatory pattern of activation. Our results suggest that T. gondii infection induces and maintains a 'proliferation response' in the infected cell which may fulfill critical growth requirements of the parasite during intracellular residence.

The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation.

Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-kappaB subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IkappaB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IkappaB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-kappaB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-kappaB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.

Host and parasite-derived IKK activities direct distinct temporal phases of NF-kappaB activation and target gene expression following Toxoplasma gondii infection.

Activation of NF-kappaB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IkappaB alpha to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IkappaB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-kappaB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-kappaB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-kappaB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-kappaB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-kappaB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.