Heparin-mediated PCR interference in SARS-CoV-2 assays and subsequent reversal with heparinase I.

Heparin is postulated to block the interaction of SARS-CoV-2 with highly glycosylated proteins which are critical for binding the angiotensin-converting enzyme 2 (ACE2), an essential mechanism for host-cell entry and viral replication. Intranasal heparin is under investigation for use as a SARS-CoV-2 preventative in the IntraNasal Heparin Trial (INHERIT, NCT05204550). Heparin directly interferes with real-time quantitative polymerase chain reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection. This study aimed to investigate the magnitude of heparin interference across various clinical laboratory testing platforms, and the reversal of any interference by degradation of heparin using the heparinase I enzyme in nasopharyngeal swab (NP) samples for SARS-CoV-2 analysis by RT-qPCR. Heparin-mediated PCR interference was evident at heparin concentrations as low as 10 IU/mL across all platforms tested, with the exclusion of the Hologic Panther Aptima SARS-CoV-2 assay. Rates of false negative or invalid results increased with increasing heparin concentrations on all platforms, except the Hologic Panther Aptima and Roche Cobas LIAT. Heparinase I reversed heparin-mediated PCR inhibition across in all samples tested, except those with initial Ct values >35. Our study shows that the use of heparin-containing nasal sprays interferes with the detection of SARS-CoV-2 in NP swab samples by RT-qPCR, a phenomenon that is not well recognised in the literature. Furthermore, this study has also demonstrated that heparin-mediated PCR inhibition can be prevented through heparinase I treatment, demonstrating restoration of clinically significant results with Ct values <35.

A placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility and differentiation impairment in syncytiotrophoblasts.

SARS-CoV-2 infection causes COVID-19. Several clinical reports have linked COVID-19 during pregnancy to negative birth outcomes and placentitis. However, the pathophysiological mechanisms underpinning SARS-CoV-2 infection during placentation and early pregnancy are not clear. Here, to shed light on this, we used induced trophoblast stem cells to generate an in vitro early placenta infection model. We identified that syncytiotrophoblasts could be infected through angiotensin-converting enzyme 2 (ACE2). Using a co-culture model of vertical transmission, we confirmed the ability of the virus to infect syncytiotrophoblasts through a previous endometrial cell infection. We further demonstrated transcriptional changes in infected syncytiotrophoblasts that led to impairment of cellular processes, reduced secretion of HCG hormone and morphological changes vital for syncytiotrophoblast function. Furthermore, different antibody strategies and antiviral drugs restore these impairments. In summary, we have established a scalable and tractable platform to study early placental cell types and highlighted its use in studying strategies to protect the placenta.

Quantifying the reduction of airborne infectious viral load using a ventilated patient hood.

BACKGROUND: Healthcare workers treating SARS-CoV-2 patients are at risk of infection by respiratory exposure to patient-emitted, virus-laden aerosols. Source control devices such as ventilated patient isolation hoods have been shown to limit the dissemination of non-infectious airborne particles in laboratory tests, but data on their performance in mitigating the airborne transmission risk of infectious viruses are lacking. AIM: We used an infectious airborne virus to quantify the ability of a ventilated hood to reduce infectious virus exposure in indoor environments. METHODS: We nebulized 109 plaque forming units (pfu) of bacteriophage PhiX174 virus into a ∼30-m3 room when the hood was active or inactive. The airborne concentration of infectious virus was measured by BioSpot-VIVAS and settle plates using plaque assay quantification on the bacterial host Escherichia coli C. The airborne particle number concentration (PNC) was also monitored continuously using an optical particle sizer. FINDINGS: The median airborne viral concentration in the room reached 1.41 × 105 pfu/m3 with the hood inactive. When active, the hood reduced infectious virus concentration in air samples by 374-fold. The deposition of infectious virus on the surface of settle plates was reduced by 87-fold. This was associated with a 109-fold reduction in total airborne particle number escape rate. CONCLUSION: A personal ventilation hood significantly reduced airborne particle escape, considerably lowering infectious virus contamination in an indoor environment. Our findings support the further development of source control devices to mitigate nosocomial infection risk among healthcare workers exposed to airborne viruses in clinical settings.

Core protocol for the adaptive Platform Trial In COVID-19 Vaccine priming and BOOsting (PICOBOO).

BACKGROUND: The need for coronavirus 2019 (COVID-19) vaccination in different age groups and populations is a subject of great uncertainty and an ongoing global debate. Critical knowledge gaps regarding COVID-19 vaccination include the duration of protection offered by different priming and booster vaccination regimens in different populations, including homologous or heterologous schedules; how vaccination impacts key elements of the immune system; how this is modified by prior or subsequent exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and future variants; and how immune responses correlate with protection against infection and disease, including antibodies and effector and T cell central memory. METHODS: The Platform Trial In COVID-19 priming and BOOsting (PICOBOO) is a multi-site, multi-arm, Bayesian, adaptive, randomised controlled platform trial. PICOBOO will expeditiously generate and translate high-quality evidence of the immunogenicity, reactogenicity and cross-protection of different COVID-19 priming and booster vaccination strategies against SARS-CoV-2 and its variants/subvariants, specific to the Australian context. While the platform is designed to be vaccine agnostic, participants will be randomised to one of three vaccines at trial commencement, including Pfizer's Comirnaty, Moderna's Spikevax or Novavax's Nuvaxovid COVID-19 vaccine. The protocol structure specifying PICOBOO is modular and hierarchical. Here, we describe the Core Protocol, which outlines the trial processes applicable to all study participants included in the platform trial. DISCUSSION: PICOBOO is the first adaptive platform trial evaluating different COVID-19 priming and booster vaccination strategies in Australia, and one of the few established internationally, that is designed to generate high-quality evidence to inform immunisation practice and policy. The modular, hierarchical protocol structure is intended to standardise outcomes, endpoints, data collection and other study processes for nested substudies included in the trial platform and to minimise duplication. It is anticipated that this flexible trial structure will enable investigators to respond with agility to new research questions as they arise, such as the utility of new vaccines (such as bivalent, or SARS-CoV-2 variant-specific vaccines) as they become available for use. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry ACTRN12622000238774. Registered on 10 February 2022.

Longitudinal evaluation of laboratory-based serological assays for SARS-CoV-2 antibody detection.

Serological assays for SARS-CoV-2 infection are now widely available for use in diagnostic laboratories. Limited data are available on the performance characteristics in different settings, and at time periods remote from the initial infection. Validation of the Abbott (Architect SARS-CoV-2 IgG), DiaSorin (Liaison SARS-CoV-2 S1/S2 IgG) and Roche (Cobas Elecsys Anti-SARS-CoV-2) assays was undertaken utilising 217 serum samples from 131 participants up to 7 months following COVID-19 infection. The Abbott and DiaSorin assays were implemented into routine laboratory workflow, with outcomes reported for 2764 clinical specimens. Sensitivity and specificity were concordant with the range reported by the manufacturers for all assays. Sensitivity across the convalescent period was highest for the Roche at 95.2-100% (95% CI 81.0-100%), then the DiaSorin at 88.1-100% (95% CI 76.0-100%), followed by the Abbott 68.2-100% (95% CI 53.4-100%). Sensitivity of the Abbott assay fell from approximately 5 months; on this assay paired serum samples for 45 participants showed a significant drop in the signal-to-cut-off ratio and 10 sero-reversion events. When used in clinical practice, all samples testing positive by both DiaSorin and Abbott assays were confirmed as true positive results. In this low prevalence setting, despite high laboratory specificity, the positive predictive value of a single positive assay was low. Comprehensive validation of serological assays is necessary to determine the optimal assay for each diagnostic setting. In this low prevalence setting we found implementation of two assays with different antibody targets maximised sensitivity and specificity, with confirmatory testing necessary for any sample which was positive in only one assay.

Symptoms and laboratory manifestations of mild COVID-19 in a repatriated cruise ship cohort.

Much of our current understanding about novel coronavirus disease 2019 (COVID-19) comes from hospitalised patients. However, the spectrum of mild and subclinical disease has implications for population-level screening and control. Forty-nine participants were recruited from a group of 99 adults repatriated from a cruise ship with a high incidence of COVID-19. Respiratory and rectal swabs were tested by polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sera were tested for anti-SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and microneutralisation assay. Symptoms, viral shedding and antibody response were examined. Forty-five participants (92%) were considered cases based on either positive PCR or positive ELISA for immunoglobulin G. Forty-two percent of cases were asymptomatic. Only 15% of symptomatic cases reported fever. Serial respiratory and rectal swabs were positive for 10% and 5% of participants respectively about 3 weeks after median symptom onset. Cycle threshold values were high (range 31-45). Attempts to isolate live virus were unsuccessful. The presence of symptoms was not associated with demographics, comorbidities or antibody response. In closed settings, incidence of COVID-19 could be almost double that suggested by symptom-based screening. Serology may be useful in diagnosis of mild disease and in aiding public health investigations.

Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew.

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Optimization of postharvest ultrasonic treatment of kiwifruit using RSM.

This study reports the optimization of ultrasonic treatment combined with sodium hypochlorite (NaOCl) solution on kiwifruit (Actinidia deliciosa) to evaluate its effect on microbial population, respiration rate and its textural quality. Response surface methodology (RSM) based on four factors three level central composite design was applied to investigate the effects of process variables on ultrasonic treatment. Four independent variables include ultrasonic intensity (184-368W/cm(2)), temperature (25-40°C), treatment time (8-15min) and concentration of the solvent (30-60ppm) were considered for this study. According to RSM analysis, the optimal treatment parameters obtained were ultrasonic intensity (368W/cm(2)), temperature (25°C), treatment time (8min) and concentration of the solvent (30ppm). Microbial population, respiration rate and some quality parameters were compared with NaOCl treated kiwifruits. An ultrasound combined with NaOCl was found to be the most effective treatment in inhibiting the microbial growth (bacteria, yeast and mold) and preserving the quality of kiwifruits, and these results suggest that the ultrasound treatment may provide an alternative for extending the shelf life of whole kiwifruit, maintains the quality of fresh cut kiwifruits and further increases the shelf life of chitosan coated fresh cut kiwifruit.

Development and Deployment of Systems-Based Approaches for the Management of Soilborne Plant Pathogens.

Biological suppression of soilborne diseases with minimal use of outside interventive actions has been difficult to achieve in high input conventional crop production systems due to the inherent risk of pest resurgence. This review examines previous approaches to the management of soilborne disease as precursors to the evolution of a systems-based approach, in which plant disease suppression through natural biological feedback mechanisms in soil is incorporated into the design and operation of cropping systems. Two case studies are provided as examples in which a systems-based approach is being developed and deployed in the production of high value crops: lettuce/strawberry production in the coastal valleys of central California (United States) and sweet basil and other herb crop production in Israel. Considerations for developing and deploying system-based approaches are discussed and operational frameworks and metrics to guide their development are presented with the goal of offering a credible alternative to conventional approaches to soilborne disease management.

Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

A model for multiseasonal spread of verticillium wilt of lettuce.

Verticillium wilt, caused by Verticillium dahliae, is a destructive disease in lettuce, and the pathogen is seedborne. Even though maximum seed infestation rates of <5% have been detected in commercial lettuce seed lots, it is necessary to establish acceptable contamination thresholds to prevent introduction and establishment of the pathogen in lettuce production fields. However, introduction of inoculum into lettuce fields for experimental purposes to determine its long term effects is undesirable. Therefore, we constructed a simulation model to study the spread of Verticillium wilt following pathogen introduction from seed. The model consists of four components: the first for simulating infection of host plants, the second for simulating reproduction of microsclerotia on diseased plants, the third for simulating the survival of microsclerotia, and the fourth for simulating the dispersal of microsclerotia. The simulation results demonstrated that the inoculum density-disease incidence curve parameters and the dispersal gradients affect disease spread in the field. Although a steep dispersal gradient facilitated the establishment of the disease in a new field with a low inoculum density, a long-tail gradient allowed microsclerotia to be dispersed over greater distances, promoting the disease spread in fields with high inoculum density. The simulation results also revealed the importance of avoiding successive lettuce crops in the same field, reducing survival rate of microsclerotia between crops, and the need for breeding resistance against V. dahliae in lettuce cultivars to lower the number of microsclerotia formed on each diseased plant. The simulation results, however, suggested that, even with a low seed infestation rate, the pathogen would eventually become established if susceptible lettuce cultivars were grown consecutively in the same field for many years. A threshold for seed infestation can be established only when two of the three drivers of the disease-(i) low microsclerotia production per diseased plant, (ii) long-tail dispersal gradient, and (iii) low microsclerotia survival between lettuce crops-are present.

Clonal expansion of Verticillium dahliae in lettuce.

Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

Comparative pathogenicity, biocontrol efficacy, and multilocus sequence typing of Verticillium nonalfalfae from the invasive Ailanthus altissima and other hosts.

Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and highly efficacious against Ailanthus and, thus, is a strong candidate for use as a biocontrol agent.

Distribution of Lettuce Big-Vein Incidence Under Three Irrigation Systems.

Spatial patterns of lettuce big-vein (LBV) incidence under furrow, sprinkler, and subsurface drip irrigation systems were determined. Because LBV pathogen is a virus and is vectored by the soilborne chytrid Olpidium brassicae, different irrigation systems likely affect the movement of the vector and were hypothesized to result in different distribution patterns and levels of the disease. Lettuce plants were mapped by recording the location of each LBV-infected or healthy plant in arbitrarily selected plots of sizes 16 by 30, 20 by 30, and 18 by 50 m in Salinas, Gonzales, and Santa Maria in California. Data were arrayed into different quadrat sizes by rearrangement, and disease incidence was calculated for each quadrat. Frequency distribution analysis and spatial autocorrelation analyses were performed on this data. LBV incidence was aggregated in all furrow-irrigated fields, four of five subsurface drip-irrigated fields, and two of three sprinkler-irrigated fields. The remaining fields had a random distribution. As the quadrat size increased, index of aggregation decreased, and vice versa. In fields under sprinkler irrigation, regardless of whether the spatial pattern of LBV was random or aggregated, no directional orientation occurred. However, under furrow or subsurface drip irrigation, the aggregation mostly occurred across the rows. Although irrigation type influenced LBV distribution pattern and incidence in lettuce fields, the differential effects of irrigation type on vector O. brassicae could not be discerned in this study. The sprinkler irrigation practiced in lettuce production until thinning may influence the vector distribution and the subsequent irrigation methods adapted for the remainder of the season in individual fields may play a significant role in disease incidence.

Aggressive angiomyxoma of the vulva in a patient with systemic lupus erythematosus.

Aggressive angiomyxoma is a rare, slow-growing mesenchymal neoplasm with a tendency to recur. It mainly involves the pelvis, vulva, perineum, vagina, and urinary bladder in adult women of reproductive age group. We describe a 26-year-old female with large swellings of both labia majora which was histologically diagnosed as aggressive angiomyxoma. She also had systemic lupus erythematosus. The swelling was surgically removed and she had no recurrence at 1-year follow-up. Although it is a rare tumor, it must be considered as a differential diagnosis for any mass in the perineum or soft tissue of the pelvis. Long-term follow-up is necessary for early diagnosis of local recurrence.

SARS CoV subunit vaccine: antibody-mediated neutralisation and enhancement.

1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).