Candida auris PCR for high-throughput infection control screening.

Candida auris has significant implications for infection control due to its multidrug resistance and spread in healthcare settings. Current culture-based screening methods are laborious and risk muco-cutaneous colonisation of laboratory staff. We describe the adaptation of a published real-time PCR for the identification of C. auris in skin swabs for high-throughput infection control screening. Two published primer and probe sets were analysed utilising serial 10-fold dilutions of 15 C. auris strains to assess the PCR limit of detection. One primer and probe set was compatible with our laboratory workflow and was selected for further development yielding a limit of detection of 1 colony forming unit per reaction. Non-C. auris isolates as well as routine skin swabs (n = 100) were tested by culture and PCR to assess specificity, where no cross-reactivity was detected. Skin swabs from a proven C. auris case (n = 6) were all both culture positive and PCR positive, while surveillance swabs from close contacts (n = 46) were all both culture negative and PCR negative. Finally, the use of a lysis buffer comprising 4 m guanidinium thiocyanate rendered swab-equivalent quantities of C. auris non-viable, providing assurance of the safety benefit of PCR over culture. The development of a PCR assay for high-throughput infection control screening is a promising method for rapid detection of C. auris with utility in an outbreak setting. LAY SUMMARY: Candida auris, a difficult to treat yeast-like fungus, has spread through healthcare facilities globally, posing a serious threat to the health of patients. We evaluated a PCR-based method suitable for screening large numbers of patient samples to rapidly and accurately detect C. auris.

Impact of Meningococcal B (4CMenB) Vaccine on Pharyngeal Neisseria meningitidis Carriage Density and Persistence in Adolescents.

BACKGROUND: Higher density of Neisseria meningitidis carriage may be associated with transmission of the meningococcus. Our aim was to establish the impact of meningococcal B (4CMenB) vaccine on N. meningitidis carriage density. METHODS: We compared 4CMenB vaccine to control among 913 South Australian students aged approximately 15-18 years in a cluster randomized trial who had N. meningitidis carriage at 12 months. Oropharyngeal swabs were collected at baseline and 12 months later to detect N. meningitidis carriage. Colony-forming units per milliliter (CFU/mL) were estimated by generating a standard curve that plotted quantitative polymerase chain reaction cycle threshold values against log-normalized CFU. RESULTS: Among the 913 students with N. meningitidis carriage at 12 months, there was no difference in mean carriage density between the vaccinated (n = 434; 3.80 log CFU/mL [standard deviation {SD}, 1.29]) and control group (n = 479; 3.73 log CFU/mL [SD, 1.30]; P = .51). Higher N. meningitidis carriage density at baseline was associated with an increase in the odds of persistent carriage at 12 months (n = 504; odds ratio [OR] per 1.0 log CFU/mL increase in density, 1.36 [95% confidence interval {CI}, 1.17-1.58]; P < .001). Students with baseline carriage who were vaccinated had decreased persistent N. meningitidis carriage at 12 months compared to unvaccinated students (81/260 [31%] vs 105/244 [43%]; OR, 0.60 [95% CI, .40-.90]; P = .01). CONCLUSIONS: 4CMenB vaccine did not reduce carriage density of N. meningitidis 12 months postvaccination, despite increased carriage clearance. Higher carriage density is likely to enable transmission through prolonged periods of population exposure. CLINICAL TRIALS REGISTRATION: NCT03089086.

Whole-Genome Sequencing of SARS-CoV-2 from Quarantine Hotel Outbreak.

Hotel quarantine for international travelers has been used to prevent coronavirus disease spread into Australia. A quarantine hotel-associated community outbreak was detected in South Australia. Real-time genomic sequencing enabled rapid confirmation tracking the outbreak to a recently returned traveler and linked 2 cases of infection in travelers at the same facility.

Intense interseasonal influenza outbreaks, Australia, 2018/19.

BackgroundInterseasonal influenza outbreaks are not unusual in countries with temperate climates and well-defined influenza seasons. Usually, these are small and diminish before the main influenza season begins. However, the 2018/19 summer-autumn interseasonal influenza period in Australia saw unprecedented large and widespread influenza outbreaks.AimOur objective was to determine the extent of the intense 2018/19 interseasonal influenza outbreaks in Australia epidemiologically and examine the genetic, antigenic and structural properties of the viruses responsible for these outbreaks.MethodsThis observational study combined the epidemiological and virological surveillance data obtained from the Australian Government Department of Health, the New South Wales Ministry of Health, sentinel outpatient surveillance, public health laboratories and data generated by the World Health Organization Collaborating Centre for Reference and Research on Influenza in Melbourne and the Singapore Agency for Science, Technology and Research.ResultsThere was a record number of laboratory-confirmed influenza cases during the interseasonal period November 2018 to May 2019 (n= 85,286; 5 times the previous 3-year average) and also more institutional outbreaks, hospitalisations and deaths, than what is normally seen.ConclusionsThe unusually large interseasonal influenza outbreaks in 2018/19 followed a mild 2018 influenza season and resulted in a very early start to the 2019 influenza season across Australia. The reasons for this unusual event have yet to be fully elucidated but are likely to be a complex mix of climatic, virological and host immunity-related factors. These outbreaks reinforce the need for year-round surveillance of influenza, even in temperate climates with strong seasonality patterns.

Coxiella burnetii dormancy in a fatal ten-year multisystem dysfunctional illness: case report.

BACKGROUND: In a previous study of a Q fever outbreak in Birmingham, our group identified a non-infective complex of Coxiella burnetii (C.b.) antigens able to survive in the host and provoked aberrant humoral and cell-mediated immunity responses. The study led to recognition of a possible pathogenic link between C.b. infection and subsequent long-term post Q fever fatigue syndrome (QFS). This report presents an unusually severe case of C.b. antigen and DNA detection in post-mortem specimens from a patient with QFS. CASE PRESENTATION: We report a 19-year old female patient who became ill with an acute unexplained febrile encephalitis-like illness, followed by increasingly severe multisystem dysfunction and death 10 years later. During life, extensive clinical and laboratory investigations from different disciplinary stand points failed to deliver a definitive identification of a cause. Given the history of susceptibility to infection from birth, acute fever and the diagnosis of "post viral syndrome", tests for infective agents were done starting with C.b. and Legionella pneumophila. The patient had previously visited farms a number of times. Comprehensive neuropathological assessment at the time of autopsy had not revealed gross or microscopic abnormalities. The aim was to extend detailed studies with the post-mortem samples and identify possible factors driving severe disturbance of homeostasis and organ dysfunction exhibited by the course of the patient's ten-year illness. Immunohistochemistry for C.b. antigen and PCR for DNA were tested on paraffin embedded blocks of autopsy tissues from brain, spleen, liver, lymph nodes (LN), bone marrow (BM), heart and lung. Standard H&E staining of brain sections was unrevealing. Immuno-staining analysis for astrocyte cytoskeleton proteins using glial fibrillary acidic protein (GFAP) antibodies showed a reactive morphology. Coxiella antigens were demonstrated in GFAP immuno-positive grey and white matter astrocytes, spleen, liver, heart, BM and LN. PCR analysis (COM1/IS1111 genes) confirmed the presence of C.b. DNA in heart, lung, spleen, liver & LN, but not in brain or BM. CONCLUSION: The study revealed the persistence of C. b. cell components in various organs, including astrocytes of the brain, in a post-infection QFS. The possible mechanisms and molecular adaptations for this alternative C.b. life style are discussed.

Direct whole-genome sequencing of HIV-1 for clinical drug-resistance analysis and public health surveillance.

BACKGROUND: Human Immunodeficiency virus type 1 (HIV-1) remains a significant global health threat partly due to its ability to develop resistance to anti-retroviral therapies. HIV-1 genotype and drug resistance analysis of the polymerase (pol) sequence is a mainstay of its clinical and public health management. However, as new treatments and resistances evolve, analysis methods must change accordingly. In this study, we outline the development and implementation of a direct whole-genome sequencing approach (dWGS) using probe-capture target-enrichment for HIV-1 genotype and drug resistance analysis. METHODS: We implemented dWGS and performed parallel pol Sanger sequencing for clinical samples, followed by comparative genotype and drug-resistance analysis. These HIV-1 WGS sequences were also utilised for a novel partitioned phylogenetic analysis. RESULTS: Optimised nucleic acid extraction and DNAse I treatment significantly increased HIV-1 whole-genome coverage and depth, and improved recovery of high-quality genomes from low viral load clinical samples, enabling routine sequencing of viral loads as low as 1000 copies/mL. Overall, dWGS was robust, accurate and more sensitive for detecting low-frequency variants at drug-resistance sites compared to Sanger sequencing. Analysis of multiple sequence regions improved phylogenetic reconstruction for recombinant HIV-1 sequences compared to analysis of pol sequence alone. CONCLUSIONS: These findings demonstrate dWGS enhances HIV-1 drug-resistance analysis by quantitative variant detection and improves reconstruction of HIV-1 phylogenies compared to traditional pol sequencing. This work supports that HIV-1 dWGS is a viable option to replace Sanger sequencing for clinical and public health applications.

The genomic epidemiology of Neisseria meningitidis carriage from a randomised controlled trial of 4CMenB vaccination in an asymptomatic adolescent population.

Background: Oropharyngeal carriage of Neisseria meningitidis is frequent during adolescence, representing a major source of invasive meningococcal disease. This study examined the impact of a serogroup B vaccination (Bexsero, GSK 4CMenB) programme on adolescent N. meningitidis carriage using genomic data. Methods: A total 34,489 oropharyngeal samples were collected as part of a state-wide cluster randomised-controlled trial in South Australia during 2017 and 2018 (NCT03089086). Samples were screened for the presence of N. meningitidis DNA by porA PCR prior to culture. Whole genome sequencing was performed on all 1772 N. meningitidis culture isolates and their genomes were analysed. Findings: Unencapsulated meningococci were predominant at baseline (36.3% of isolates), followed by MenB (31.0%), and MenY (20.5%). Most MenB were ST-6058 from hyperinvasive cc41/44, or ST-32 and ST-2870 from cc32. For MenY, ST-23 and ST-1655 from cc23 were prevalent. Meningococcal carriage was mostly unchanged due to the vaccination programme; however, a significant reduction in ST-53 capsule-null meningococci prevalence was observed in 2018 compared to 2017 (OR = 0.52; 95% CI: 0.30-0.87, p = 0.0106). This effect was larger in the vaccinated compared to the control group (OR = 0.37; 95% CI: 0.12-0.98, p = 0.0368). Interpretation: While deployment of the 4CMenB vaccination did not alter the carriage of hyperinvasive MenB in the vaccinated population, it altered the carriage of other N. meningitidis sequence types following the vaccination program. Our findings suggest 4CMenB vaccination is unlikely to reduce transmission of hyperinvasive N. meningitidis strains and therefore ongoing targeted vaccination is likely a more effective public health intervention. Funding: This work was funded by GlaxoSmithKline Biologicals SA.

From Biowaste to Lab-Bench: Low-Cost Magnetic Iron Oxide Nanoparticles for RNA Extraction and SARS-CoV-2 Diagnostics.

The gold standard for diagnostics of SARS-CoV-2 (COVID-19) virus is based on real-time polymerase chain reaction (RT-PCR) using centralized PCR facilities and commercial viral RNA extraction kits. One of the key components of these kits are magnetic beads composed of silica coated magnetic iron oxide (Fe2O3 or Fe3O4) nanoparticles, needed for the selective extraction of RNA. At the beginning of the pandemic in 2019, due to a high demand across the world there were severe shortages of many reagents and consumables, including these magnetic beads required for testing for SARS-CoV-2. Laboratories needed to source these products elsewhere, preferably at a comparable or lower cost. Here, we describe the development of a simple, low-cost and scalable preparation of magnetic nanoparticles (MNPs) from biowaste and demonstrate their successful application in viral RNA extraction and the detection of COVID-19. These MNPs have a unique nanoplatelet shape with a high surface area, which are beneficial features, expected to provide improved RNA adsorption, better dispersion and processing ability compared with commercial spherical magnetic beads. Their performance in COVID-19 RNA extraction was evaluated in comparison with commercial magnetic beads and the results presented here showed comparable results for high throughput PCR analysis. The presented magnetic nanoplatelets generated from biomass waste are safe, low-cost, simple to produce in large scale and could provide a significantly reduced cost of nucleic acid extraction for SARS-CoV-2 and other DNA and RNA viruses.

Evaluation of the cobas 4800 CT/NG test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae.

OBJECTIVES: To investigate the performance of the fully automated cobas 4800 CT/NG test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. METHODS: The study was conducted using 900 clinical specimens (496 urine and 404 swab specimens) for C trachomatis testing, of which 498 specimens (318 urine and 180 swab specimens) were also tested for N gonorrhoeae. The results of the cobas 4800 CT/NG test were compared with those obtained from the Roche COBAS AMPLICOR CT/NG and COBAS TaqMan CT assays. N gonorrhoeae-positive specimens were further tested using in-house, real-time PCR assays. A panel of 223 Neisseria isolates was used to further investigate the performance of the cobas 4800 N gonorrhoeae assay. RESULTS: For urine specimens, the sensitivity, specificity and negative and positive predictive values of the cobas 4800 CT/NG test were 94.5%, 99.5%, 98.8% and 97.7%, respectively, for C trachomatis, and 92.9%, 100%, 99.7% and 100%, respectively, for N gonorrhoeae. For swab specimens, the sensitivity, specificity and negative and positive predictive values were 92.0%, 100%, 99.5% and 100%, respectively, for C trachomatis, and 100%, 99.4%, 100% and 90.0%, respectively, for N gonorrhoeae. All N gonorrhoeae isolates were positive and all non-gonococcal Neisseria strains were negative by the cobas 4800 N gonorrhoeae assay. CONCLUSIONS: The cobas 4800 CT/NG test is suitable for high through-put identification of C trachomatis and N gonorrhoeae infections.