Haycocknema perplexum is a rare and emerging cause of parasitic myositis. Detection and surveillance are of growing importance given the increasing degree of climate conditions conducive to transmission. In this study, we developed a real-time polymerase chain reaction (PCR) method for direct detection of two genomic regions of H. perplexum. The real-time PCR assays, SSU-PCR and COX-1-PCR, targeted the small subunit of nuclear ribosomal RNA and cytochrome oxidase-1 genomic regions, respectively. The performance of the assays was assessed using a panel of H. perplexum samples, both fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue (n=22, derived from eight patients) and tissue biopsy specimens not derived from H. perplexum (n=8). Both H. perplexum assays showed 92% and 84% sensitivity for SSU and COX-1 targets, respectively, 100% specificity, and negative and positive predictive values of 100% and 93%, respectively. The results indicate that the limit of detection was 10-5 dilution [cycle threshold (Ct) value 40.2] for COX-1-PCR, and 10-3 dilution (Ct value 39.6) for SSU-PCR, making the latter a more sensitive assay for detecting lower concentrations of organism in the patient biopsy. The sensitivity of fresh frozen samples was superior to FFPE samples. All but one sample was negative following treatment. Feasibility of real-time PCR detection of H. perplexum directly from tissue biopsies has been demonstrated for diagnosis, possible test of cure, and could enhance transmission surveillance.

ABSTRACT Digital surface‐enhanced Raman scattering (SERS) immunoassays digitize epitope binding events to achieve ultrasensitive protein detection. However, existing implementations predominantly rely on single‐epitope recognition, yielding 1D molecular view of antigen structure and obscuring how conformational heterogeneity or mutation‐induced changes affect epitopes. This limitation cannot be resolved simply by combining multiple monoclonal antibodies, as heterogeneous conjugation and uncontrolled binding collapse epitope‐specific responses into pooled, non‐assignable signals. Here, we introduce EpiCount‐SERS (Epitope‐Resolved Digital Counting by SERS), a multi‐epitope digital SERS framework that enables epitope‐resolved immunochemical profiling. The platform employs nanobody‐based bispecific antibody fragments that pair epitope‐specific nanobodies with a unified anti‐methoxy polyethylene glycol conjugation domain, enabling orientation‐controlled attachment to spectrally encoded SERS nanotags. Epitope‐specific binding events are discretized into independent digital channels, allowing distinct epitopes on the SARS‐CoV‐2 receptor‐binding domain to be interrogated in parallel. Digital enumeration across these channels generates epitope‐resolved molecular fingerprints that capture information beyond single‐epitope assays. EpiCount‐SERS achieves sub‐ng mL − 1 sensitivity for recombinant protein, detects inactivated virus at approximately 10 2 copies µL − 1 , and classifies clinical nasopharyngeal swab samples with accuracy of 88.3% (area under the curve = 0.9467). Because epitope binders can be exchanged without altering the unified conjugation strategy, EpiCount‐SERS provides a scalable framework for digital immunochemical profiling of structurally dynamic protein targets.

Surface-enhanced Raman scattering (SERS) immunoassays are powerful analytical tools for protein detection while typically rely on the availability of suitable antibodies. Compared to full-length antibodies, antibody fragments provide advantages such as rapid and cost-effective production. However, oriented conjugation of antibody fragments to SERS nanotags, essential for maintaining their functionality, remains relatively under-explored. Here, we introduce a bispecific antibody (BsAb)-programmable digital immunochemistry detection regime implemented on an advanced digital SERS platform ("DigibiSERS"), enabling deterministic molecular orientation, variance-robust single-particle event calling, and kinetic regularization within one unified platform. Specifically, we fuse an anti-nucleocapsid nanobody with a single-chain variable fragment targeting methoxy polyethylene glycol (mPEG) grafted onto the SERS nanotag surfaces. This design facilitates straightforward, oriented BsAb conjugation, preserving its functionality. The DigibiSERS platform, incorporating single-particle active SERS nanotags, nanomixing-enhanced microchips, and digital readouts, demonstrates high sensitivity and specificity, achieving detection limits of 2.01 ng/mL for nucleocapsid protein and 2.7 copies/mL for virus. An area under the curve (AUC) of 0.8783 highlights the potential of engineered antibody fragments in enhancing the clinical sensitivity and practicality of SERS-based immunoassays for infectious disease diagnostics.

Using metatranscriptomics, we identified Trubanaman virus in cerebrospinal fluid from a severely immunocompromised man who died of encephalitis in Queensland, Australia. Virus sequences were related to orthobunyaviruses previously detected in mosquitoes in Australia. Testing for other causes yielded negative results, suggesting that Trubanaman virus was the cause of this fatal encephalitis case.

Chlamydia trachomatis causes sexually transmitted urogenital infections, as well as ocular infections, the latter mostly spread between children and responsible for the blinding disease trachoma. The strains causing these two types of infection are generally distinct, but there is some evidence that urogenital strains can infect the eye and cause conjunctivitis, possibly indicating transmission on fingers of adults or adolescents to children. In what we believe is the first study of its kind in a setting of high prevalence of both urogenital and ocular infection, we characterized C. trachomatis strains from 107 ocular and 95 urine samples collected through cross-sectional population surveys in Nauru, employing household-based sampling. Ocular samples were collected during a national baseline prevalence survey in 2019 in children aged 1-9 years. Urine samples were collected from adults participating in a national survey before (March 2020) and after (December 2020) a national mass drug administration (MDA) of azithromycin for trachoma in April 2020. We used a cluster survey design to recruit participants, with clusters based on Nauru's districts and a predetermined number of households randomly selected in each cluster. There was no attempt to recruit from the same households across the three survey rounds. Only genotype C, recognized as an ocular strain, was identified in eye specimens from children, whereas six urogenital genotypes (D, E, G, J, Ja, and L1) were detected in adult urine samples, with genotype D most frequent. We, therefore, concluded that, in this highly endemic setting for both urogenital and ocular infection with C. trachomatis, there were two distinct networks of transmission with no evidence of crossover, at least at the population level.IMPORTANCEChlamydia infections are a public health issue with two broad manifestations: ocular infections, mostly found in children, and sexually transmitted infections of the genital tract and anus that can lead to adverse reproductive health outcomes. While generally caused by different C. trachomatis strains, there is some evidence that strains considered to be predominantly sexually transmitted can infect the conjunctiva and cause signs resembling trachoma. Possible strain crossover has raised concern about whether eye infection with genital C. trachomatis strains confers a drive toward visual impairment and blindness, and the potential for such infections to lead to overestimates of trachoma prevalence. In what we believe to be the first study of its kind, we identified distinct ocular strains in pediatric ocular swabs and urogenital strains in adult urine specimens in Nauru and concluded that urogenital C. trachomatis strains are not contributing to ocular disease despite the high prevalence of urogenital chlamydia.

Surface-enhanced Raman scattering (SERS) immunoassays have emerged as highly sensitive, multiplexed analytical techniques for detecting protein biomarkers. Traditional SERS immunoassays typically rely on antibody-based SERS probes for target protein detection; however, it is challenging to obtain antibodies that are both highly effective at identifying natural proteins and suitable for SERS probe conjugation. Herein, we engineer a MultiValent Probe (MVP), consisting of multivalent nanobodies as the protein-targeting ligand to provide improved binding avidity and Raman reporter-coated gold-silver alloy nanoboxes for single-particle signal readouts. The multivalent nanobodies exhibit precise antigen recognition and exceptional affinity, and are expressed in a mammalian system for cost-effective and large-scale production. We thoroughly characterize the MVP via nanoparticle tracking analysis, nanoflow cytometry, and differential centrifugal sedimentation. To further enhance assay performance, we integrate MVP with a nanomixing-enhanced microfluidic chip to develop an MVP-based SERS-microfluidic immunoassay. As a proof of concept, we demonstrate the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins and virions from multiple strains in clinical nasopharyngeal samples (39 healthy and 39 infected), showing 84.6% concordance with RT-qPCR. This work highlights the potential of MVP-incorporated SERS-microfluidic immunoassays for diagnostics of pandemic diseases and broader applications in detecting a wide range of viral pathogens.

Haycocknema perplexum is a rare and emerging cause of parasitic myositis. Detection and surveillance are of growing importance given the increasing degree of climate conditions conducive to transmission. In this study, we developed a real-time PCR method for direct detection of two genomic regions of Haycocknema perplexum . The real-time PCR assays, SSU-PCR and COX1-PCR targeted the small subunit of nuclear ribosomal RNA and cytochrome oxidase-1 genomic regions, respectively. The performance of the assays was assessed using a panel of H. perplexum samples, both fresh frozen and formalin fixed paraffin embedded tissue (FFPE) (n = 22, derived from eight patients) and non- H. perplexum (n = 8) tissue biopsy specimens. Both H. perplexum assays showed 83% sensitivity and 100% specificity, with negative and positive predictive values of 100% and 93% respectively. The results indicate that the LOD was 10 −5 dilution (C t value 40.2) for COX1-PCR, and 10 −3 dilution (C t value 39.6) for SSU-PCR, making the latter a more sensitive assay for detecting lower concentrations of organism in the patient biopsy. The sensitivity of fresh frozen samples was superior to FFPE samples. All but 1 sample was negative following treatment. Feasibility of real-time PCR detection of H. perplexum directly from tissue biopsies has been demonstrated for diagnosis, possible test of cure and could enhance transmission surveillance.

The continued emergence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires ongoing genetic surveillance to support public health responses. The expansion of reliable next generation sequence (NGS) platforms has enabled the rapid characterisation of the constant emergence of new SARS-CoV-2 variants using nasopharyngeal swab specimens. Several studies have assessed the ability of COVIDSeq to type earlier SARS-CoV-2 strains (pre-Delta) rapidly and successfully, however, there is limited data showing suitability against Omicron variants. In the present study, we evaluated the performance of the Illumina COVIDSeq Assay as a streamlined amplicon-based NGS platform for detection and typing of Omicron variants. Our results demonstrate the high performance of SARS-CoV-2 sequencing using the COVIDSeq approach, with good repeatability, reproducibility and sensitivity for samples approaching CT 31. The COVIDSeq approach was 100% concordant with samples previously characterized by sequencing methods. The quick library preparation process and high throughput kit made it ideal for reflex testing, with a total time required for sequencing and analysis of approximately two days. This study demonstrates the effectiveness and versatility of the amplicon-based NGS characterisation method for SARS-CoV-2, providing a foundation for further research and development of custom-designed amplicon panels targeting different microorganisms.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

Background: Cytomegalovirus (CMV) is a viral infection which establishes lifelong latency, often reactivating and causing disease in immunosuppressed individuals, including haematopoietic stem cell transplant (HSCT) re-cipients. Treatment can be problematic due to antiviral resistance which substantially increases the risk of pa-tient mortality. Diagnostic testing capabilities for CMV antiviral resistance in Australia and elsewhere have traditionally relied on gene-specific Sanger sequencing approaches, however, are now being superseded by next generation sequencing protocols.Objective: Provide a snapshot of local mutations and explore the feasibility of the ViroKey?(R) SQ FLEX Genotyping Assay (Vela Diagnostics Pty Ltd) by examining sequencing success.Method: Performed sequencing on adult (n = 38) and paediatric (n = 81) plasma samples, over a large range of viral loads (above and below the assay recommended threshold of >1,000 International Units (IU)/mL; noting most of our paediatric samples have loads <1,000 IU/mL).Results: Eleven test runs (including three repeat runs; 14 to 15 samples per run) were conducted, and four runs were deemed valid. The overall individual sample success rate for the four evaluable test runs was 71.2% (42/59 samples); 80.4% (37/46) samples >1,000 IU/mL were valid. Ten clinically important antiviral resistance mu-tations were detected, the most common being A594V in the UL97 gene, found in 6 (5%) samples.Conclusions: A range of technical issues were experienced, however with improvement this platform could be a useful addition to routine pathology workflows, providing timely antiviral resistance results for patients un-dergoing HSCT.

Cytomegalovirus (CMV) is a ubiquitous virus which causes a mild illness in healthy individuals. In immunocompromised individuals, such as children receiving haematopoietic stem cell transplantation, CMV can reactivate, causing serious disease and increasing the risk of death. CMV can be effectively treated with antiviral drugs, but antiviral resistance is an increasingly common complication. Available therapies are associated with adverse effects such as bone marrow suppression and renal impairment, making the choice of appropriate treatment challenging. New agents are emerging and require evaluation in children to establish their role. This review will discuss established and emerging diagnostic tools and treatment options for CMV, including antiviral resistant CMV, in children undergoing haematopoietic stem cell transplant.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold–silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/μL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

The continuous transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required that diagnostic capabilities be constantly monitored and updated as new variants emerge and prior variants disappear. Although whole genome sequencing provides full characterisation of SARS-CoV-2 directly from patient samples, this has limited throughput and requires sufficient resources. To enhance screening for circulating variants, we designed a rapid in-house RT-PCR assay to target a spike mutation (D950N) in Delta variants, which is not detected in the remaining variants of concern (VOCs). Assay sensitivity for detecting Delta variants was 93% and specificity was 100% using a sequenced sample bank of several lineages. As the D950N mutation is prevalent in > 95% of the global Delta variant sequences deposited in GISAID, this assay has the potential to provide rapid results to determine if the samples are presumptively Delta variants and can support clinicians in timely clinical decision-making for effective treatments and surveillance.

An ongoing outbreak of syphilis in Australia, first reported in the state of Queensland in 2011, has led to increasing cases of congenital syphilis, including several deaths. Here, we applied multi-locus sequence typing (MLST) on available Treponema pallidum PCR-positive samples from the state of Queensland from the beginning of the outbreak to July 2020. In total, 393 samples from 337 males and 56 females were genotyped. Of 36 different Treponema pallidum sequence types (ST) observed, the two most common STs, ST 1 (also reported to be a dominant strain in various other countries) and ST 100 (the latter differing from ST 1 by only one single nucleotide polymorphism (SNP) based on the MLST scheme), together comprised 69% (271/393) of all samples, including the majority of samples in females (79%; 44/56). ST 1 was prevalent throughout the entire study period. Both strains remained the most common STs during the year 2020 where social distancing and other measures were implemented due to the COVID-19 pandemic. Both STs had high male-to-female ratios and included male rectal infections, therefore suggestive of occurrence primarily among men-who-have-sex-with-men (MSM). Hence, bridging from MSM to heterosexual networks may potentially contribute to infections among females, but further studies are needed to confirm this. Overall, there was considerable diversity of Treponema pallidum genotypes observed throughout the study period, but the fact that two key strains accounted for the majority of infections, including among females, stresses the need for further investigations into the transmission of these strains, and potentially a need for targeted public health interventions to better control the spread of syphilis in Queensland.

Background Mycoplasma genitalium was recently added to the CDC’s antimicrobial resistance threats ‘watch list’, as it has rapidly become resistant to mainstay treatments. In Australia, treatment failure with fluoroquinolones remain commonplace, even when Sanger sequencing fails to identify evidence of resistance mutations. Methods Suspecting that Sanger sequencing may miss low-load mixed infections, we applied three additional PCR-based approaches (allele-specific primer-based PCR, probe-based PCR and amplicon deep sequencing) to detect mutations associated with fluoroquinolone susceptibility/resistance. We focused on resistance mutations at amino acid positions 83 and 87 of parC, as these were previously shown to be common in Australia. Results Our results showed evidence of mixtures of fluoroquinolone-susceptible and -resistant strains in up to 27/423 samples (6.4%). These included 1 sample that was indicated to be mixed by Sanger sequencing and all three additional PCR methods, 6 samples detected by two or more of the additional PCRs but not by Sanger sequencing and finally 20 samples that were detected by only one of the additional PCR methods. A key question was whether Sanger sequencing failed to detect fluoroquinolone resistance in any samples; overall, we observed that Sanger sequencing failed to detect fluoroquinolone resistance in up to 3.8% (16/423) of samples. Conclusions The presence of mixed susceptibility infections may have important implications for clinical patient management and stresses the need for appropriate detection of resistance and selection of antimicrobials to ensure appropriate treatment of M. genitalium infections.

Although the tropical disease, dengue, has been studied exhaustedly for over 60 years, we are no closer to possessing a safe and protective vaccine to prevent the estimated 390 million infections yearly. Dengue virus, which causes fever, joint and muscle pain, vomiting, and skin rash, mutates rapidly to remain a continuous biological threat to half the world’s population. The overall complexities of the human immune response, together with the constantly evolving virus, make it a difficult disease to target effectively with a vaccine. Since 2017, we have been investigating a novel therapy using Defective Interfering virus Particles (DIPs), which are naturally produced by the virus during infection. We have found that these small particles can interfere, and thereby reduce, parental virus replication with the hope that the host clears infection earlier for a better disease outcome. We aim to identify and investigate the mode of action of these particles for therapeutic capability.

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.