Characterizing the acute antibody response of monkeypox and MVA-BN vaccine following an Australian outbreak.

In response to the emergence of the monkeypox virus (MPXV) in Australia in May 2022, we developed and evaluated indirect immunofluorescence assays (IFA) for MPXV and Vaccinia virus (VACV) IgG and IgM antibodies using serum samples from patients with nucleic acid amplification test (NAAT)-confirmed mpox and uninfected unvaccinated controls. Additionally, 47 healthcare workers receiving two doses of the third-generation smallpox vaccine Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) undertook serial serum collection to describe the serological response to vaccination. MPXV antibodies were detected in 16/18 individuals with NAAT-confirmed mpox (sensitivity 0.89, specificity 1.00), and VACV antibodies were detected in 28/29 individuals who received two doses of MVA-BN vaccine (sensitivity 0.97, specificity 1.00). Detectable antibody in subjects historically vaccinated with early-generation vaccines against smallpox was found in 7/7 subjects, at a median of 48 years following vaccination. MPXV NAAT-positive patients with serum samples collected within the first 14 days after rash onset had detectable IgG and IgM in 9/12 and 5/12 of patients, respectively, with maintenance of IgG and disappearance of IgM titers after 60 days. While specificity was high when testing unvaccinated and uninfected subjects, significant cross-reactivity between MPXV and VACV antibodies was observed.

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.

In Phi29-α-hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.

3D microelectrode arrays, pushing the bounds of sensitivity toward a generic platform for point-of-care diagnostics.

The increased sensitivity of microelectrode arrays (MEAs) over macroelectrodes for biosensing is well established, and results from reducing the diffusion gradient of the target species to and from the electrode surfaces. The current study describes the fabrication and characterisation of a polymer-based MEA, which exploits the advantages of three dimensionality (3D). Firstly, the unique 3D formfactor promotes release of the gold tips from an inert layer in a controlled fashion, to form a highly reproducible array of microelectrodes in a single step. The 3D topography of the fabricated MEAs significantly enhances the diffusion profile of the target species to the electrode which results in higher sensitivity. Furthermore, the "sharpness" of the 3D structure induces differential current distribution that is focused at the apices of the individual electrodes, reducing the active area, and thereby overcoming the requirement for the electrodes to be sub-micron in size before true MEA behaviour can be achieved. The electrochemical characteristics of the 3D MEAs shows ideal micro-electrode behaviour, with a level of sensitivity of three orders of magnitude greater than that of enzyme-linked immunosorbent assays (ELISA), as the optical based gold standard. The application of the 3D MEAs uses the combination of enzyme-label and substrate approach employed in ELISAs as a generic basis for biosensing and can hence be applied to the plethora of targets that utilise the ELISA approach. As an example, the 3D MEAs are applied to the detection of RNA and demonstrate a level of detection down to single digit picomolar concentrations.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

Hybrid bilayer membranes as platforms for biomimicry and catalysis.

Hybrid bilayer membrane (HBM) platforms represent an emerging nanoscale bio-inspired interface that has broad implications in energy catalysis and smart molecular devices. An HBM contains multiple modular components that include an underlying inorganic surface with a biological layer appended on top. The inorganic interface serves as a support with robust mechanical properties that can also be decorated with functional moieties, sensing units and catalytic active sites. The biological layer contains lipids and membrane-bound entities that facilitate or alter the activity and selectivity of the embedded functional motifs. With their structural complexity and functional flexibility, HBMs have been demonstrated to enhance catalytic turnover frequency and regulate product selectivity of the O2 and CO2 reduction reactions, which have applications in fuel cells and electrolysers. HBMs can also steer the mechanistic pathways of proton-coupled electron transfer (PCET) reactions of quinones and metal complexes by tuning electron and proton delivery rates. Beyond energy catalysis, HBMs have been equipped with enzyme mimics and membrane-bound redox agents to recapitulate natural energy transport chains. With channels and carriers incorporated, HBM sensors can quantify transmembrane events. This Review serves to summarize the major accomplishments achieved using HBMs in the past decade.

Experimental study of a string-based counterflow wet electrostatic precipitator for collection of fine and ultrafine particles.

Wet electrostatic precipitators (WESP) have been widely studied for collecting fine and ultrafine particles, such as diesel particulate matter (DPM), which have deleterious effects on human health. Here, we report an experimental and numerical simulation study on a novel string-based two-stage WESP. Our new design incorporates grounded vertically aligned polymer strings, along which thin films of water flow down. The water beads, generated by intrinsic flow instability, travel down the strings and collect charged particles in the counterflowing gas stream. We performed experiments using two different geometric configurations of WESP: rectangular and cylindrical. We examined the effects of the WESP electrode bias voltage, air stream velocity, and water flow rate on the number-based fractional collection efficiency for particles of diameters ranging from 10 nm to 2.5 µm. The collection efficiency improves with increasing bias voltages or decreasing airflow rates. At liquid-to-gas (L/G) as low as approximately 0.0066, our design delivers a collection efficiency over 70% even for fine and ultrafine particles. The rectangular and cylindrical configurations exhibit similar collection efficiencies under nominally identical experimental conditions. We also compare the water-to-air mass flow rate ratio, air flow rate per unit collector volume, and collection efficiency of our string-based design with those of previously reported WESPs. The present work demonstrates a promising design for a highly efficient, compact, and scalable two-stage WESPs with minimal water consumption.Implications: Wet Electrostatic Precipitators (WESPs) are highly effective for collecting fine particles in exhaust air streams from various sources such as diesel engines, power plants, and oil refineries. However, their large-scale adoption has been limited by high water usage and reduced collection efficiencies for ultrafine particles. We perform experimental and numerical investigation to characterize the collection efficiency and water flow rate-dependence of a new design of WESP. The string-based counterflow WESP reported in this study offers number-based collection efficiencies >70% at air flow rates per collector volume as high as 4.36 (m3/s)/m3 for particles of diameters ranging from 10 nm - 2.5 µm, while significantly reducing water usage. Our work provides a basis for the design of more compact and water-efficient WESPs.

Cell-based Culture Informs Infectivity and Safe De-Isolation Assessments in Patients with Coronavirus Disease 2019.

BACKGROUND: The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA by reverse-transcription polymerase chain reaction (PCR) does not necessarily indicate shedding of infective virions. There are limited data on the correlation between the isolation of SARS-CoV-2, which likely indicates infectivity, and PCR. METHODS: A total of 195 patients with Coronavirus disease 2019 were tested (outpatients, n = 178; inpatients, n = 12; and critically unwell patients admitted to the intensive care unit [ICU] patients, n = 5). SARS-CoV-2 PCR-positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2-induced CPE was confirmed by PCR of culture supernatant. Where no CPE was observed, PCR was performed on day 4 to confirm absence of virus replication. The cycle thresholds (Cts) of the day 4 PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined where Ctsample - Ctculture was ≥3. RESULTS: Of 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was isolated from 56 (24%), including in 28 of 181 (15%), 19 of 42 (45%), and 9 of 11 samples (82%) collected from outpatients, inpatients, and ICU patients, respectively. All 56 samples had Ctsample ≤32; CPE was observed in 46 (20%). The mean duration from symptom onset to culture positivity was 4.5 days (range, 0-18). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (P < .001) and ICU patients (P < .0001) compared with outpatients, and in samples with lower Ctsample. CONCLUSIONS: SARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.

Group B streptococcal disease and genotypes in Australian infants.

AIMS: Estimate the incidence, risk factors and clinical features of group B streptococcal (GBS) disease in Australian infants and compare bacterial genotypes between infant disease and maternal carriage. METHODS: The Australian Paediatric Surveillance Unit conducted a study of invasive GBS disease in infants aged 0-90 days between July 2005 and June 2008. Clinical data were obtained by questionnaire. GBS isolates from affected infants and antenatal carriers were referred for genotyping. RESULTS: One hundred fifty confirmed cases were reported: 88 early-onset (EOD; 0-6 days) and 62 late-onset disease (LOD). Based on review of selected laboratory records, they represented ∼1/3 of all cases. Estimated national EOD and LOD rates were 0.38 and 0.19/1000 live births, respectively. Admission to intensive care was common (44%), and 7% of infants died. One or more obstetric indications for intrapartum antibiotic prophylaxis (IAP) were identified in 51% of mothers; 53% of mothers were screened for GBS carriage, and screening was positive in 45%; only 25% of women with clinical or microbiological risk factors were given IAP (no significant differences between EOD and LOD groups). Distribution of GBS genotypes differed significantly between invasive and vaginal isolates: virulent serosubtype III-2/sequence type 17 was strongly associated with LOD but uncommon among EOD and vaginal isolates. CONCLUSIONS: Estimated GBS disease rates remain relatively low despite poor predictive values of clinical and microbiological criteria for, and compliance with, IAP. Clinical and microbiological epidemiology of LOD differs significantly from that of EOD. Further reduction in infant morbidity and mortality from GBS disease will require new strategies.

Identification of surface proteins of group B streptococci: serotyping versus genotyping.

We compared serotyping to genotyping of group B streptococcal (GBS) surface proteins in 147 Australasian isolates. Results were concordant for the two methods in 73.8% of 122 isolates, discordant for three and partially discordant for 29 isolates. For the purpose of epidemiological typing of GBS, genotyping is superior to serotyping.

Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes. RESULTS: It was found that a SNP set derived from the MLST database on the basis of maximization of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. CONCLUSION: A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.

Use of phenotypic and molecular serotype identification methods to characterize previously nonserotypeable group B streptococci.

Among 1,762 isolates of Streptococcus agalactiae (group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning cpsH to cpsM). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsG region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the cpsE-cpsF-cspG region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516; P < 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.

Production of activated carbons from waste tire--process design and economical analysis.

The process design and economic analysis of process plants to produce activated carbons from waste tires and coal have been performed. The potential range of products from each process has been considered, namely for waste tire--pyro-gas, active carbon, carbon black and pyro-oil; for coal--pyro-gas and active carbons. Sensitivity analyses have been carried out on the main process factors; these are product price, production capacity, total production cost, capital investment and the tipping fee. Net present values for the two plants at various discount factors have been determined and the internal rates of return have been determined as 27.4% and 18.9% for the waste tire plant and the coal plant, respectively.

Film and intraparticle mass transfer during the adsorption of metal ions onto bone char.

The sorption of three metal ions, namely, copper, cadmium, and zinc, onto bone char has been studied in terms of equilibrium and rate studies. Equilibrium studies have been analyzed using the Langmuir isotherm equation and the maximum sorption capacities for the metals were 0.477, 0.709, and 0.505 mmolg(-1) bone char for cadmium, copper, and zinc ions, respectively. The kinetic experimental data were used to analyze the effect of external film boundary layer and intraparticle mass transfer resistance on the sorption process and its significance. Four methods of determining the external film transport coefficient were developed and tested; three utilized experimental data to obtain the coefficient and the fourth method was completely empirical. The three experimentally based models give very similar results and consequently similar values of the deviation error values, whereas the error values for the empirical correlation were greater than these three values. The results also demonstrated that the methods for determining the film coefficient could be integrated into more complex diffusion-transport models such as film-intraparticle diffusion processes.

Sorption equilibria of metal ions on bone char.

The ability of bone char to adsorb three metal ions, namely, copper(II), zinc(II) and cadmium(II) ions from wastewater has been studied. Three single-component equilibrium systems and three binary equilibrium systems have been measured experimentally. The three single-component equilibrium data were analyzed using the Langmuir and the Sips equilibrium isotherm equations. The Sips isotherm gave a better fit of the experimental data than the Langmuir isotherm based on the sum of squares errors (SSE) analysis. The Cu-Zn, Cu-Cd and Cd-Zn binary equilibrium experimental data were examined by incorporating the Langmuir and the Sips isotherm equations into the ideal adsorbed solution theory (IAST). The solution methods and the predicted results for the three binary systems at different metal ion compositions have been evaluated. In addition, the application of the IAST to the model prediction for the fixed bed system is presented.