Embryo mortality in a captive-bred, Critically Endangered amphibian.

The Critically Endangered southern corroboree frog Pseudophryne corroboree is dependent upon captive assurance colonies for its continued survival. Although the captive breeding programme for this species has largely been successful, embryonic mortality remains high (40-90% per year). This study aimed to investigate the causes of mortality in P. corroboree embryos in the captive collection at Melbourne Zoo. During the 2021 breeding season, we investigated 108 abnormal embryos to determine the impact of infections and anatomical deformities on survival and used culture and molecular methods to identify microbes. Overall, 100% of abnormal embryos had fungal infections, and of these, 41.6% also had anatomical deformities. The mortality rate in abnormal embryos was 89.8%; however, we detected no difference in survival in any of the 3 observed fungal growth patterns or between deformed and non-deformed embryos. Sanger sequencing of the ITS region identified fungal isolates belonging to the genus Ilyonectria, the first record in a vertebrate host, and another as a Plectosphaerella sp., which is the first record of infection in an embryo. Dominant bacteria identified were of the genera Herbaspirillum and Flavobacterium; however, their role in the mortality is unknown. Fungal infection and deformities have a significant impact on embryo survival in captive-bred P. corroboree. In a species which relies on captive breeding, identifying and reducing the impacts of embryonic mortality can inform conservation efforts and improve reintroduction outcomes.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Candidaemia and a risk predictive model for overall mortality: a prospective multicentre study.

BACKGROUND: Candidaemia is associated with high mortality. Variables associated with mortality have been published previously, but not developed into a risk predictive model for mortality. We sought to describe the current epidemiology of candidaemia in Australia, analyse predictors of 30-day all-cause mortality, and develop and validate a mortality risk predictive model. METHODS: Adults with candidaemia were studied prospectively over 12 months at eight institutions. Clinical and laboratory variables at time of blood culture-positivity were subject to multivariate analysis for association with 30-day all-cause mortality. A predictive score for mortality was examined by area under receiver operator characteristic curves and a historical data set was used for validation. RESULTS: The median age of 133 patients with candidaemia was 62 years; 76 (57%) were male and 57 (43%) were female. Co-morbidities included underlying haematologic malignancy (n = 20; 15%), and solid organ malignancy in (n = 25; 19%); 55 (41%) were in an intensive care unit (ICU). Non-albicans Candida spp. accounted for 61% of cases (81/133). All-cause 30-day mortality was 31%. A gastrointestinal or unknown source was associated with higher overall mortality than an intravascular or urologic source (p < 0.01). A risk predictive score based on age > 65 years, ICU admission, chronic organ dysfunction, preceding surgery within 30 days, haematological malignancy, source of candidaemia and antibiotic therapy for ≥10 days stratified patients into < 20% or ≥ 20% predicted mortality. The model retained accuracy when validated against a historical dataset (n = 741). CONCLUSIONS: Mortality in patients with candidaemia remains high. A simple mortality risk predictive score stratifying patients with candidaemia into < 20% and ≥ 20% 30-day mortality is presented. This model uses information available at time of candidaemia diagnosis is easy to incorporate into decision support systems. Further validation of this model is warranted.

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.

OBJECTIVES: Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata. METHODS: The antifungal susceptibility of 12 clinical isolates and one ATCC strain of C. glabrata was determined using the Sensititre YeastOne® YO10 assay. These included three isolate pairs where the second isolate of each pair had developed a rise in drug MICs. Single nucleotide polymorphisms (SNPs) in genes known to be linked to echinocandin, azole and 5-fluorocytosine resistance were analysed in all isolates through NGS. RESULTS: High-quality non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgCDR1, CgPDR1 and FCY2 were identified. For two of three isolate pairs, there was a >60-fold rise in MICs to all echinocandins in the second isolate from each pair; one echinocandin-resistant isolate harboured a mutation in FKS1 (S629P) and the other in FKS2 (S663P). Of the third pair, both the 5-fluorocytosine-susceptible, and resistant isolates had a mutation in FCY2 (A237T). SNPs in CgPDR1 were found in pan-azole-resistant isolates. SNPs in other genes linked to azole resistance (CgCDR1, ERG9 and CgFLR1) were present in both azole-susceptible and azole-resistant isolates. SNPs were also identified in Candida adhesin genes EPA1, EPA6, PWP2 and PWP5 but their presence was not associated with higher drug MICs. CONCLUSIONS: Genome-wide analysis of antifungal resistance markers was feasible and simultaneously revealed mutation patterns of genes implicated in resistance to different antifungal drug classes.

Mucormycosis in Australia: contemporary epidemiology and outcomes.

Mucormycosis is the second most common cause of invasive mould infection and causes disease in diverse hosts, including those who are immuno-competent. We conducted a multicentre retrospective study of proven and probable cases of mucormycosis diagnosed between 2004-2012 to determine the epidemiology and outcome determinants in Australia. Seventy-four cases were identified (63 proven, 11 probable). The majority (54.1%) were caused by Rhizopus spp. Patients who sustained trauma were more likely to have non-Rhizopus infections relative to patients without trauma (OR 9.0, p 0.001, 95% CI 2.1-42.8). Haematological malignancy (48.6%), chemotherapy (42.9%), corticosteroids (52.7%), diabetes mellitus (27%) and trauma (22.9%) were the most common co-morbidities or risk factors. Rheumatological/autoimmune disorders occurred in nine (12.1%) instances. Eight (10.8%) cases had no underlying co-morbidity and were more likely to have associated trauma (7/8; 87.5% versus 10/66; 15.2%; p <0.001). Disseminated infection was common (39.2%). Apophysomyces spp. and Saksenaea spp. caused infection in immuno-competent hosts, most frequently associated with trauma and affected sites other than lung and sinuses. The 180-day mortality was 56.7%. The strongest predictors of mortality were rheumatological/autoimmune disorder (OR = 24.0, p 0.038 95% CI 1.2-481.4), haematological malignancy (OR = 7.7, p 0.001, 95% CI 2.3-25.2) and admission to intensive care unit (OR = 4.2, p 0.02, 95% CI 1.3-13.8). Most deaths occurred within one month. Thereafter we observed divergence in survival between the haematological and non-haematological populations (p 0.006). The mortality of mucormycosis remains particularly high in the immuno-compromised host. Underlying rheumatological/autoimmune disorders are a previously under-appreciated risk for infection and poor outcome.

Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: a single center experience.

The role of panfungal polymerase chain reaction (PCR) assays for diagnosis of invasive fungal disease (IFD) is inadequately defined. We describe the use of an internal transcribed spacer 1 (ITS-1) region-directed panfungal PCR in this context at a tertiary referral transplant center. A retrospective review of patients at Alfred Health, Melbourne, Australia (2009-2014) who had clinical samples referred for panfungal PCR testing was conducted. Baseline patient characteristics, antifungal drug history, fungal culture/histopathology, and radiology results were recorded. For bronchoalveolar lavage (BAL) fluid samples, identification of a fungus other than a Candida spp. was defined as a potential pathogen.Of 138 panfungal PCR tests (108 patients), 41 (30%) were positive for a fungal product. Ninety-seven percent (134/138) of specimens were from immunocompromised hosts. Thirteen percent (19/138) of panfungal PCR positive results were for potential pathogens and potential pathogens were detected more frequently in tissue as compared with BAL (12/13 vs. 6/26; P = .0001). No positive panfungal PCR results were obtained from CSF specimens. If histopathology examination was negative, panfungal PCR identified a potential pathogen in only 12% (11/94) of specimens. For the 20 culture negative/histopathology positive specimens, diagnosis of IFD to causative species level by panfungal PCR occurred in 35% (6/20).Sterile site specimens, in particular tissue, were more frequently panfungal PCR positive for potential pathogens than BAL. The utility of panfungal PCR appears greatest in tissue specimens, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. Based on the results of this study we are now only testing tissue specimens by panfungal PCR.

Microsphaeropsis arundinis skin and soft tissue infection in renal transplant recipients: three case reports and a review of the literature.

Microsphaeropsis arundinis, a dematiaceous mold, is emerging as a cause of skin and soft tissue infection in immunocompromised hosts. Diagnosis is challenging because of the difficulty in identifying Microsphaeropsis species morphologically and few data are available to guide optimal management. We report 3 renal transplant recipients with M. arundinis soft tissue infection, where the etiological agent was diagnosed using DNA sequencing, and who were successfully treated with prolonged courses of extended-spectrum triazole antifungal agents.

Invasive infections due to filamentous fungi other than Aspergillus: epidemiology and determinants of mortality.

The epidemiology of invasive fungal disease (IFD) due to filamentous fungi other than Aspergillus may be changing. We analysed clinical, microbiological and outcome data in Australian patients to determine the predisposing factors and identify determinants of mortality. Proven and probable non-Aspergillus mould infections (defined according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria) from 2004 to 2012 were evaluated in a multicentre study. Variables associated with infection and mortality were determined. Of 162 episodes of non-Aspergillus IFD, 145 (89.5%) were proven infections and 17 (10.5%) were probable infections. The pathogens included 29 fungal species/species complexes; mucormycetes (45.7%) and Scedosporium species (33.3%) were most common. The commonest comorbidities were haematological malignancies (HMs) (46.3%) diabetes mellitus (23.5%), and chronic pulmonary disease (16%); antecedent trauma was present in 21% of cases. Twenty-five (15.4%) patients had no immunocompromised status or comorbidity, and were more likely to have acquired infection following major trauma (p <0.01); 61 (37.7%) of cases affected patients without HMs or transplantation. Antifungal therapy was administered to 93.2% of patients (median 68 days, interquartile range 19-275), and adjunctive surgery was performed in 58.6%. The all-cause 90-day mortality was 44.4%; HMs and intensive-care admission were the strongest predictors of death (both p <0.001). Survival varied by fungal group, with the risk of death being significantly lower in patients with dematiaceous mould infections than in patients with other non-Aspergillus mould infections. Non-Aspergillus IFD affected diverse patient groups, including non-immunocompromised hosts and those outside traditional risk groups; therefore, definitions of IFD in these patients are required. Given the high mortality, increased recognition of infections and accurate identification of the causative agent are required.

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

Sinonasal and sino-orbital aspergillosis in 23 cats: aetiology, clinicopathological features and treatment outcomes.

Aetiology, clinicopathological findings and treatment outcomes were documented in 23 cats (1.5-13 years of age) with sinonasal (SNA, n=6) or sino-orbital (SOA, n=17) aspergillosis. Cases recruited retrospectively and prospectively were included if fungal hyphae were identified on cytological or histological examination and the fungal pathogen was identified by PCR and DNA sequencing (ITS1 or ITS1-5.8S-ITS2 regions, rDNA gene cluster). Fungal culture was positive in 22/23 cases. In cases of SNA, the fungal pathogen was Aspergillus fumigatus (n=4), Neosartorya fischeri or A. lentulus (n=1) or a non-speciated Neosartorya spp. (n=1). In all cases of SOA (n=17), the fungal pathogen was identified as Neosartorya spp. Nine cats had brachycephalic conformation. Cats with SNA were more likely to be infected with A. fumigatus and had a better prognosis than cats with SOA.

Clinician response to Candida organisms in the urine of patients attending hospital.

The epidemiology of 54 episodes of candiduria with respect to clinical risk factors, species of Candida and physician response to the isolation of Candida in urine were studied in an observational survey over 3 months. Candida spp. were isolated from 4.7% of positive urine cultures. Common predisposing conditions included antibiotic use (74.1%), urinary drainage devices (57.4%), surgery (51.9%), intensive care unit (ICU) or high-dependency care unit (HDU) admission (42.6%) and urinary tract (UT) disease (18.5%). Upper UT infection was uncommon (n = 3). Of 65 Candida isolates, C. albicans predominated (85.2%), followed by C. glabrata (27.8%) and other Candida spp. (6.2%). All isolates were susceptible to fluconazole, itraconazole, voriconazole, amphotericin and caspofungin. Indwelling urinary catheters were removed in 76.2% of episodes. Antifungal therapy was initiated in 33.3% of cases independently of patient symptoms, underlying disease or Candida colony count. Patients in ICU/HDUs were significantly more likely to receive antifungal agents than those outside these units (p < 0.001). Fluconazole was the most common drug prescribed (77.8%). Clearance of candiduria occurred independently of antifungal therapy (p = 0.60). Physicians often did not follow up a positive urine result for Candida. Efforts to increase clinician awareness of current recommendations for managing candiduria and further study to elucidate specific risk factors in defined patient populations are warranted.

Ulocladium atrum keratitis.

We report a case of Ulocladium atrum keratitis in a 43-year-old man. No predisposing event was known. He received natamycin and fluconazole drops and the infection resolved. The isolate was identified by morphological and rRNA gene sequence analyses. U. atrum is a dematiaceous hyphomycete not hitherto reported to infect humans.

Clonal reproduction and limited dispersal in an environmental population of Cryptococcus neoformans var gattii isolates from Australia.

Cryptococcus neoformans var. gattii is a causative agent of cryptococcosis and is thought to have a specific ecological association with a number of Eucalyptus species in Australia. However, the role that the tree plays in the life cycle of the fungus and the nature of the infectious propagule are not well understood. This study set out to examine whether sexual recombination is occurring in a natural population of C. neoformans var. gattii and whether the fungus disseminates between colonized trees. Thirty C. neoformans var. gattii isolates, consisting of both the alpha and a mating types, were collected from 13 Eucalyptus camaldulensis trees growing along a riverbank in Renmark, South Australia. The genetic diversity within the population was studied by using amplified fragment length polymorphism fingerprinting, and each isolate was assigned a unique multilocus genotype. Population genetic analyses of the multilocus data found no evidence of genetic exchange between members of the population, indicating a clonal population structure. Canonical variate analysis was then used to study the relationship between isolates from different colonized trees. Isolates from individual trees were strongly correlated, and it appeared that dispersal between trees was not occurring to any appreciable extent. These results suggest that the eucalypt may not be the primary niche for C. neoformans var. gattii but that the decaying wood present in hollows on these trees may provide a favorable substrate for extensive clonal propagation of the yeast cells.

Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia.

Cryptococcus neoformans var. gattii lives in association with certain species of eucalyptus trees and is a causative agent of cryptococcosis. It exists as two mating types, MATalpha and MATa, which is determined by a single-locus, two-allele system. In the closely related C. neoformans var. neoformans, the alpha mating type has been found to outnumber its a counterpart by at least 30:1, but there have been very limited data on the proportions of each mating type in C. neoformans var. gattii. In the present study, specific PCR primers were designed to amplify two separate alpha-mating-type genes from C. neoformans var. gattii strains. These were used to survey for the presence of the two mating types in clinical and environmental collections of C. neoformans var. gattii strains from Australia. Sixty-eight of 69 clinical isolates produced both alpha mating type-specific bands and were assumed to be of the alpha mating type. The majority of environmental isolates were also of the alpha mating type, but the a mating type was located in two separate areas. In one area, the a mating type outnumbered the alpha mating type by 27:2, but in the second area, the ratio of the two mating types was close to the 50:50 ratio expected for sexual recombination.

Characterization of bile salt uptake by Giardia lamblia.

We have shown previously that Giardia lamblia takes up conjugated bile salt in vitro, and have now investigated the mechanism by which this occurs. Uptake of sodium taurocholate (TC) and glycocholate (GC) with respect to time had an initial exponential component followed by a linear component, consistent with a combination of both active and passive transport processes. The presence of an active transport process was further supported by experiments which showed that bile salt uptake: (i) was concentration dependent (apparent Km's for TC and GC were 0.21 and 0.63 mM, respectively); (ii) was competitively inhibitable; (iii) was reduced by the metabolic inhibitor sodium fluoride (50 mM) and low temperature (4 degrees C). Bile salt was not taken up by glutaraldehyde-fixed parasites, indicating that bile salt was not merely being adsorbed on to the parasite surface. Differential centrifugation of lyzed parasites following exposure to radiolabelled GC, showed that the majority of bile salt was located in the cytosol fraction (76%) with a relatively minor component associated with cell membrane, indicating that bile salt had been internalized. Bile salt analysis of extracts of parasites and culture medium indicated that GC had not been metabolized by Giardia. Thus, like the mammalian ileum, Giardia appears to take up conjugated bile salts by active and passive transport processes. Conjugated bile salts are known to promote encystation and thus these uptake mechanisms may constitute an important survival mechanism for the parasite enabling it to complete its life cycle.

The pharmacology of fluparoxan: a selective alpha 2-adrenoceptor antagonist.

1. This paper describes the pharmacology of the novel alpha 2-adrenoceptor antagonist fluparoxan (GR 50360) which is currently being studied clinically as a potential anti-depressant. Idazoxan and yohimbine were included in many studies for comparison. 2. In the rat isolated, field-stimulated vas deferens and the guinea-pig isolated, field-stimulated ileum preparations, fluparoxan was a reversible competitive antagonist of the inhibitory responses to the alpha 2-adrenoceptor agonist UK-14304 with pKB values of 7.87 and 7.89 respectively. In the rat isolated anococcygeus muscle, fluparoxan was a much weaker competitive antagonist of the contractile response to the alpha 1-adrenoceptor agonist phenylephrine with a pKB of 4.45 giving an alpha 2: alpha 1-adrenoceptor selectivity ratio of greater than 2500. 3. In the conscious mouse, fluparoxan (0.2-3.0 mg kg-1) was effective by the oral route and of similar potency to idazoxan in preventing clonidine-induced hypothermia and antinociception. In the rat, UK-14304-induced hypothermia (ED50 = 1.4 mg kg-1, p.o. or 0.5 mg kg-1, i.v.) and rotarod impairment (ED50 = 1.1 mg kg-1 p.o. or 1.3 mg kg-1, i.v.) were antagonized by fluparoxan. Fluparoxan, 0.67-6 mg kg-1, p.o., also prevented UK-14304-induced sedation and bradycardia in the dog. 4. In specificity studies fluparoxan had low or no affinity for a wide range of neurotransmitter receptor sites at concentrations up to at least 1 x 10(-5) M. It displayed weak affinity for 5-HT1A (pIC50 = 5.9) and 5-HT1B (pKi = 5.5) binding sites in rat brain. 5. We conclude that fluparoxan is a highly selective and potent alpha 2-adrenoceptor antagonist. The density of rat brain [3H]-dihydroalprenolol binding sites was reduced by 26% when fluparoxan was administered chronically for 6 days at a dose of 12 mg kg- 1 orally twice daily. The down-regulation of beta-adrenoceptors by fluparoxan is consistent with its antidepressant potential.