Serosurvey for Japanese encephalitis virus antibodies following an outbreak in an immunologically naïve population, Victoria, 2022: a cross-sectional study.

OBJECTIVES: To investigate the distribution and prevalence of Japanese encephalitis virus (JEV) antibody (as evidence of past infection) in northern Victoria following the 2022 Japanese encephalitis outbreak, seeking to identify groups of people at particular risk of infection; to investigate the distribution and prevalence of antibodies to two related flaviviruses, Murray Valley encephalitis virus (MVEV) and West Nile virus Kunjin subtype (KUNV). STUDY DESIGN: Cross-sectional serosurvey (part of a national JEV serosurveillance program). SETTING: Three northern Victorian local public health units (Ovens Murray, Goulburn Valley, Loddon Mallee), 8 August - 1 December 2022. PARTICIPANTS: People opportunistically recruited at pathology collection centres and by targeted recruitment through community outreach and advertisements. People vaccinated against or who had been diagnosed with Japanese encephalitis were ineligible for participation, as were those born in countries where JEV is endemic. MAIN OUTCOME MEASURES: Seroprevalence of JEV IgG antibody, overall and by selected factors of interest (occupations, water body exposure, recreational activities and locations, exposure to animals, protective measures). RESULTS: 813 participants were recruited (median age, 59 years [interquartile range, 42-69 years]; 496 female [61%]); 27 were JEV IgG-seropositive (3.3%; 95% confidence interval [CI], 2.2-4.8%) (median age, 73 years [interquartile range, 63-78 years]; 13 female [48%]); none were IgM-seropositive. JEV IgG-seropositive participants were identified at all recruitment locations, including those without identified cases of Japanese encephalitis. The only risk factors associated with JEV IgG-seropositivity were age (per year: prevalence odds ratio [POR], 1.07; 95% CI, 1.03-1.10) and exposure to feral pigs (POR, 21; 95% CI, 1.7-190). The seroprevalence of antibody to MVEV was 3.0% (95% CI, 1.9-4.5%; 23 of 760 participants), and of KUNV antibody 3.3% (95% CI, 2.1-4.8%; 25 of 761). CONCLUSIONS: People living in northern Victoria are vulnerable to future JEV infection, but few risk factors are consistently associated with infection. Additional prevention strategies, including expanding vaccine eligibility, may be required to protect people in this region from Japanese encephalitis.

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection.

Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Designed for a pandemic: Mitigating the risk of SARS-CoV-2 transmission through hospital design and infrastructure.

BACKGROUND: To describe the new Royal Adelaide Hospital (RAH) design and infrastructure features that helped mitigate the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission within the hospital during the pre-vaccination and pre-antiviral period. METHOD: The RAH infrastructure, design and initial pandemic response was assessed. A retrospective review of all confirmed or suspected coronavirus disease 2019 (COVID-19) patients admitted from 1 February 2020 to 30 May 2020 was also performed to assess risk of transmission. Outbreak response reports were reviewed to identify episodes of nosocomial COVID-19. RESULTS: Key infrastructure features include single-bed overnight rooms with dedicated bathrooms, creation of pandemic areas accessible only to pandemic staff, and sophisticated air-handling units with improved ventilation. A total of 264 COVID-19 related admission occurred, with 113 confirmed cases and 1579 total cumulative bed days. Despite a limited understanding of SARS-CoV-2 transmission, no vaccination or anti-viral therapy, global shortages of particulate filter respirators and restricted testing during this period, only one probable nosocomial COVID-19 case occurred in a healthcare worker, with no nosocomial cases involving patients. CONCLUSIONS: The RAH design and pandemic features complimented existing infection control interventions and was important in limiting nosocomial spread of SARS-CoV-2.

Use of a point-of-care test to rapidly assess levels of SARS-CoV-2 nasal neutralising antibodies in vaccines and breakthrough infected individuals.

Despite SARS-CoV-2 vaccines eliciting systemic neutralising antibodies (nAbs), breakthrough infections still regularly occur. Infection helps to generate mucosal immunity, possibly reducing disease transmission. Monitoring mucosal nAbs is predominantly restricted to lab-based assays, which have limited application to the public. In this multi-site study, we used lateral-flow surrogate neutralisation tests to measure mucosal and systemic nAbs in vaccinated and breakthrough infected individuals in Australia and Singapore. Using three lateral flow assays to detect SARS-CoV-2 nAbs, we demonstrated that nasal mucosal nAbs were present in 71.4 (95% CI 56.3-82.9%) to 85.7% (95% CI 71.8-93.7%) of individuals with breakthrough infection (positivity rate was dependent upon the type of test), whereas only 20.7 (95% CI 17.1-49.4%) to 34.5% (95% CI 19.8-52.7%) of vaccinated individuals without breakthrough infection had detectible nasal mucosal nAbs. Of the individuals with breakthrough infection, collective mucosal anti-S antibody detection in confirmatory assays was 92.9% (95% CI 80.3-98.2%) of samples, while 72.4% (95% CI 54.1-85.5%) of the vaccinated individuals who had not experienced a breakthrough infection were positive to anti-S antibody. All breakthrough infected individuals produced systemic anti-N antibodies; however, these antibodies were not detected in the nasal cavity. Mucosal immunity is likely to play a role in limiting the transmission of SARS-CoV-2 and lateral flow neutralisation tests provide a rapid readout of mucosal nAbs at the point-of-care.

Laboratory evaluation of ELISA and indirect immunofluorescence assay in response to emergence of Japanese encephalitis virus genotype IV in Australia.

The unexpected recent emergence of Japanese encephalitis virus (JEV) genotype IV in multiple southern states of Australia necessitated an evaluation of JEV serological tests suitable for diagnosing acute infection and for seroprevalence studies. This study examined the analytical and clinical performance of two high-throughput JEV assays, Euroimmun immunofluorescence assay (IFA) and Euroimmun enzyme-linked immunosorbent assay (ELISA), across four cohorts; (1) surveillance of piggery workers in outbreak areas, (2) surveillance of residents in outbreak areas, (3) acute JEV infection and (4) post-JEV vaccination. ELISA and IFA IgM demonstrated minimal cross-reactivity (0-1.8%) with other endemic flaviviruses, with high sensitivity (100%) for acute JEV infection in this low endemicity setting. Differences in IgG serodynamics between the two assays suggest convalescent and paired testing with IgM are critical in diagnosing acute infection. High assay concordance was observed between ELISA and IFA when used in serosurveillance (97.4% agreement, Cohen' κ 0.74 [95% CI 0.614-0.860]) and vaccination cohorts (91.1% agreement, Cohen's κ 0.806 [95% CI 0.672-0.941]). In conclusion, this study highlights the clinical & epidemiological applications and limitations of these two commercial JEV assays.

Assessment of HBV infection and inter-host cellular responses using intrahepatic cholangiocyte organoids.

Intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral, and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma derived HBV (genotype B & C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, cccDNA, extracellular DNA), and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg, and HBsAg. Donor dependent drug-responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor dependent variation in viral dynamics and cellular responses. These features can be utilized for development of personalized drug testing platform for antivirals.

Laboratory assessment of a multi-target assay for the rapid detection of viruses causing vesicular diseases.

BACKGROUND: The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. OBJECTIVE: The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. RESULTS: The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. CONCLUSION: The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses.

Evaluation of 16 molecular assays for the detection of orthopox and mpox viruses.

BACKGROUND: The current global mpox virus (MPXV) outbreak has been declared a Public Health Emergency of International Concern by WHO, with more than 80,000 cases confirmed across multiple continents. Diagnosis is confirmed by PCR of viral DNA from vesicle and other swabs. OBJECTIVE: The aim of this study was to assess commercial RT PCR assays for Orthopoxvirus (OPX) and MPXV for analytical sensitivity, and percent agreements and compare them to primer/probe sets employed at the Victorian Infectious Diseases Reference Laboratory (VIDRL), Centers for Disease Control andPrevention (CDC) and US Army Medical Research Institute of Infectious Diseases (USAMRIID). Limits of detection (LOD), intra-run variability, cross-reactivity and performance on forty clinical samples was assessed on eleven commercial assays and five primer/probe combinations used at VIDRL, CDC and USAMRIID. RESULTS: All assays were able to detect OPX and MPXV (LOD 57 to 14,495 copies/mL) with intra-run coefficients of variation between Cycle thresholds of 0.58 and 3.44, and there was no unexpected cross-reactivity. All assays demonstrated 100% negative percent agreement with clinical samples and all but one yielded 100% positive percent agreement. CONCLUSIONS: Variations in LOD between assays may be dependent on the platform used and sample type. Despite the overall comparable performance of the assays assessed, it is important that routine laboratories perform in-house validations before implementing RT PCR for OPX and/or MPXV as reliable and accurate laboratory diagnosis of MPXV and isolation is crucial to containing the spread of this current outbreak and informing public health interventions and response.

Correlation between monkeypox viral load and infectious virus in clinical specimens.

BACKGROUND: In the 2022 mpox outbreak, several studies have explored longitudinal DNA shedding of mpox virus (MPXV) using PCR. However, there are fewer studies assessing infectivity in cell culture, and, by inference, MPXV transmissibility. Such information could help inform infection control and public health guidelines. AIMS AND METHODS: The aim of this study was to correlate cell culture infectivity of clinical samples with viral loads in clinical samples. Between May to October 2022, clinical samples from different body sites sent to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for MPXV PCR detection were cultured in Vero cells as a surrogate for infectivity. RESULTS: In the study period, 144 samples from 70 patients were tested by MPXV PCR. Viral loads in skin lesions were significantly higher than those in throat or nasopharyngeal samples (median Ct 22.0 vs 29.0, p = 0.0013 and median Ct 22.0 vs 36.5, p = 0.0001, respectively). Similarly, viral loads were significantly higher in anal samples compared to throat or nasopharyngeal samples (median Ct 20.0 vs. 29.0, p=<0.0001 and median Ct 20.0 vs. 36.5, p=<0.0001, respectively). Viral culture was successfully performed in 80/94 samples. Using logistic regression analysis, 50% of the samples were positive in viral culture at Ct 34.1 (95% confidence intervals 32.1-37.4). CONCLUSIONS: Our data further validate recent findings showing that samples with a higher MPXV viral load are more likely to demonstrate infectivity in cell culture. Although the presence of infectious virus in cell culture may not directly translate with clinical transmission risk, our data may be used as an adjunct help inform guidelines on testing and isolation policies in individuals with mpox.

Mpox diagnostics: Review of current and emerging technologies.

Mpox is a zoonotic disease caused by monkeypox virus (MPXV) from the Orthopoxvirus genus. Unprecedented transmission events have led to more than 70 000 cases reported worldwide by October 2022. The change in mpox epidemiology has raised concerns of its ability to establish endemicity beyond its traditional geographical locations. In this review, we discuss the current understanding of mpox virology and viral dynamics that are relevant to mpox diagnostics. A synopsis of the traditional and emerging laboratory technologies useful for MPXV detection and in guiding "elimination" strategies is outlined in this review. Importantly, development in MPXV genomics has rapidly advanced our understanding of the role of viral evolution and adaptation in the current outbreak.

Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates.

Background: Rapid identification of mycobacteria has been made possible with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) in recent years. Working with high concentrations of mycobacteria in a PC-3 containment facility makes MALDI-TOF cumbersome and costly. Therefore removing the inactivated isolate's protein extract from the PC-3 facility is needed for efficient identification in a routine PC-2 laboratory. Methods: This work describes a novel chemical and mechanical disruption protein extraction method, which provides reliable MALDI-TOF results from solid and liquid media, while ensuring laboratory safety. Results: When compared to sequencing results, 93.9% of the clinical isolates were identified in LJ media and 89% of the clinical isolates were identified in MGIT media. Conclusion: The MIPE protocol produces a high quality protein extract with improved isolate identification without compromising result turn-around-times or laboratory safety.

First human case of fatal Halicephalobus gingivalis meningoencephalitis in Australia.

Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool.