First experience of implementing Candida auris real-time PCR for surveillance in the UK: detection of multiple introductions with two international clades and improved patient outcomes.

BACKGROUND: Candida auris has been associated with rapid transmission and high mortality. A novel PCR-based surveillance programme was initiated at a London teaching hospital from January 2018. The results of this implementation until March 2019 are presented along with the clinical, transmission and phylogenetic characteristics encountered in that setting. METHODS: A real-time PCR assay for C. auris was developed, validated, and implemented for direct use on skin swabs and urine. Environmental swabs were also tested by PCR as an emergency outbreak-control measure. Clinical risk factors and outcomes of patients were determined. Environmental dispersal was assessed using 24 h settle plate cultures around nine colonized patients followed by air sampling around one colonized patient during high- and low-turbulence activities. Sequencing was performed using Illumina HiSeq and maximum likelihood phylogenies were constructed using rapid bootstrap analysis. RESULTS: Twenty-one C. auris colonized patients were identified. Median turnaround time of colonization detection reduced from 141 h (5.8 days) to approximately 24 h enabling rapid infection-control precautions. Settle plates detected 70-600 cfu/m2 around colonized patients over 24 h and air sampling suggested dispersal during turbulent activities. C. auris DNA was detected from 35.7% environmental swabs. Despite being in a high-risk setting, no patients developed invasive infection. Sequencing analysis of isolates from this centre identified two introductions of the South Asian (Clade I) and one of the South African (Clade III) strain. CONCLUSION: The PCR offers a rapid, scalable method of screening and supports clinical risk reduction in settings likely to encounter multiple introductions.

Bronchoalveolar lavage fluid IMMY Sona Aspergillus lateral-flow assay for the diagnosis of invasive pulmonary aspergillosis: a prospective, real life evaluation.

Prompt and reliable diagnosis of invasive pulmonary aspergillosis (IPA) is essential for early initiation of antifungal therapy. We evaluated bronchoalveolar lavage (BAL) fluid IMMY Sona Aspergillus lateral-flow assay (IMMY LFA) in 92 individuals with suspected pulmonary infection. Sensitivity and specificity (vs. host factor but no IPA) of BAL IMMY LFA for diagnosis of IPA in individuals with any European Organisation for Research and Treatment of Cancer-defied "host factor" were 67% and 85%, respectively. Performance appeared better in individuals with renal transplantation (100%, 100%), compared to those with hematological malignancy and/or allogenic stem cell transplantation (70%, 78%). We found BAL IMMY LFA to be a convenient and useful addition to our diagnostic armory for IPA. LAY ABSTRACT: We evaluated a new test for diagnosing invasive pulmonary aspergillosis from bronchoscopy samples. We tested 92 people and found that it was 67% sensitive and 85% specific (compared to diagnosis according to a set of internationally recognised criteria). We found this test convenient and useful.

Yeasticidal activity of chemical disinfectants and antiseptics against Candida auris.

Candida auris appears to be transmitted readily between patients, yet information regarding the efficacy of environmental disinfection and skin decolonization is lacking. A quantitative suspension test (EN 13624:2013) was used to evaluate the yeasticidal activity of different chemical disinfectants and antiseptics against C. auris and Candida albicans. When tested in suspension, both a chlorine-based disinfectant and iodine-based skin antiseptic were effective against C. auris, suggesting that their use could reduce environmental contamination and skin colonization, respectively, if applied appropriately. Chlorhexidine-based products may also be effective. However, in this study, activity depended on formulation, specifically the presence of isopropyl alcohol.

Pseudotumoral presentation of fungating mycetoma caused by Phaeoacremonium fuscum in a renal transplant patient.

Eumycetoma is an unusual infection in immunocompromised patients outside the tropics, caused by a variety of fungal pathogens. We describe the case of a 51-year-old renal transplant recipient who presented with a large pseudotumoral foot lesion necessitating complete surgical excision of the lesion. Cultures and molecular diagnosis confirmed Phaeoacremonium fuscum. This is the first case, to our knowledge, of fungating mycetoma caused by this fungal species in a solid organ transplant recipient.

First confirmation of Pseudogymnoascus destructans in British bats and hibernacula.

White-nose syndrome (WNS) is a fatal fungal infection of bats in North America caused by Pseudogymnoascus destructans. P. destructans has been confirmed in Continental Europe but not associated with mass mortality. Its presence in Great Britain was unknown. Opportunistic sampling of bats in GB began during the winter of 2009. Any dead bats or samples from live bats with visible fungal growths were submitted to the Animal Health and Veterinary Laboratories Agency for culture. Active surveillance by targeted environmental sampling of hibernacula was carried out during the winter of 2012/2013. Six hibernacula were selected by their proximity to Continental Europe. Five samples, a combination of surface swabs or sediment samples, were collected. These were sent to the Center for Microbial Genetics and Genomics, Northern Arizona University, for P. destructans PCR. Forty-eight incidents were investigated between March 2009 and July 2013. They consisted of 46 bat carcases and 31 other samples. A suspected P. destructans isolate was cultured from a live Daubenton's bat (Myotis daubentonii) sampled in February 2013. This isolate was confirmed by the Mycology Reference Laboratory, Bristol (Public Health England), as P. destructans. A variety of fungi were isolated from the rest but all were considered to be saprophytic or incidental. P. destructans was also confirmed by the Center for Microbial Genetics and Genomics in five of the six sites surveyed.

Isolation of the fungus Geosmithia argillacea in sputum of people with cystic fibrosis.

We report the repeated isolation of the fungus Geosmithia argillacea from sputum samples of people with cystic fibrosis. Identification was based on morphology and DNA sequence analysis. Isolation of G. argillacea did not appear to be associated with clinical deterioration. The pathogenic potential of G. argillacea is discussed.

Adiaspiromycosis mimicking widespread malignancy in a patient with pulmonary adenocarcinoma.

Adiaspiromycosis, a mycotic disease of small animals, has rarely been reported in humans. The principle causative organism in Europe is Emmonsia crescens. Inhaled, dust-borne spores of E crescens fail to germinate in host tissue, instead increasing in size dramatically to become thick-walled, round adiapsores, which induce a granulomatous response. The pathological effects depend on the spore burden and host immunocompetence, and range from asymptomatic infection through to necrogranulomatous pneumonia, respiratory failure and, rarely, death. Diagnosis is principally made through histological examination. This report describes a case of a patient with low-grade, localised pulmonary adenocarcinoma with occult adiaspiromycosis that radiologically mimicked widespread malignancy. It is believed to be the first reported human case of adiaspiromycosis in the UK.

Domain II hairpin structure in ITS1 sequences as an aid in differentiating recently evolved animal and plant pathogenic fungi.

The hypothesis that ITS structural features can be used to define fungal groups, where sequence analysis is unsatisfactory, was examined in plant and animal pathogenic fungi. Structural models of ITS1 regions were predicted for presumed closely related species in Colletotrichum and Trichophyton anamorphs of Arthroderma species. Structural alignment of models and comparison with ITS sequence analysis identified a variable region in a conserved hairpin formed from a common inverted repeat. Thirteen different hairpin structure models were obtained for Colletotrichum species and five different models were obtained for Trichophyton species. The different structure types could be matched to individual species and species complexes as defined by ITS sequence analysis.

Sub-cutaneous phaeohyphomycosis caused by Cladophialophora devriesii in a United Kingdom resident.

An 83-year-old diabetic man receiving corticosteroids developed a forearm lesion. Histology confirmed the presence of a dematiaceous fungus, with associated granulomatous inflammation. Culture of a biopsy yielded fungal colonies with branching chains of single-celled, melanised, dry, sympodial conidia, which were identified as Cladophialophora devriesii on the basis of morphology and rDNA gene sequencing. To date, C. devriesii has been a relatively rare cause of human disease. To our knowledge, this is only the second case to be described, and the first report of infection in a UK resident.

Detailed analysis of the requirements of hepatitis A virus internal ribosome entry segment for the eukaryotic initiation factor complex eIF4F.

The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation.

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.