Catriona Halliday

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

ABSTRACT Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n = 38) or solely histologically proven (n = 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n = 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.

Galactomannan and PCR versus culture and histology for directing use of antifungal treatment for invasive aspergillosis in high-risk haematology patients: a randomised controlled trial

BACKGROUND Empirical treatment with antifungal drugs is often used in haematology patients at high risk of invasive aspergillosis. We compared a standard diagnostic strategy (culture and histology) with a rapid biomarker-based diagnostic strategy (aspergillus galactomannan and PCR) for directing the use of antifungal treatment in this group of patients. METHODS In this open-label, parallel-group, randomised controlled trial, eligible patients were adults undergoing allogeneic stem-cell transplantation or chemotherapy for acute leukaemia, with no history of invasive fungal disease. Enrolled patients were randomly assigned (1:1) by a computer-generated schedule to follow either a standard diagnostic strategy (based on culture and histology) or a biomarker-based diagnostic strategy (aspergillus galactomannan and PCR) to direct treatment with antifungal drugs. Patients, were followed up for 26 weeks or until death. Masking of the use of different diagnostic tests was not possible for patients, treating physicians, or investigators. The primary endpoint was empirical treatment with antifungal drugs in the 26 weeks after enrolment (for the biomarker-based diagnostic strategy, a single postive galactomannan or PCR result was deemed insufficient to confirm invasive aspergillosis, so treatment in this context was classified as empirical). This outcome was assessed by an independent data review committee from which the study allocations were masked. Analyses were by intention to treat and included all enrolled patients. This study is registered with ClinicalTrial.gov, number NCT00163722. FINDINGS 240 eligible patients were recruited from six Australian centres between Sept 30, 2005, and Nov 19, 2009. 122 were assigned the standard diagnostic strategy and 118 the biomarker-based diagnostic strategy. 39 patients (32%) in the standard diagnosis group and 18 (15%) in the biomarker diagnosis group received empirical antifungal treatment (difference 17%, 95% CI 4-26; p=0·002). The numbers of patients who had hepatotoxic and nephrotoxic effects did not differ significantly between the standard diagnosis and biomarker diagnosis groups (hepatotoxic effects: 21 [17%] vs 12 [10%], p=0·11; nephrotoxic effects: 52 [43%] vs 60 [51%], p=0·20). INTERPRETATION Use of aspergillus galactomannan and PCR to direct treatment reduced use of empirical antifungal treatment. This approach is an effective strategy for the management of invasive aspergillosis in high-risk haematology patients. FUNDING Australian National Health and Medical Research Council, Cancer Council New South Wales, Pfizer, Merck, Gilead Sciences.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Identification of Yeasts Is Contingent on Robust Reference Spectra

Background Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods. Methods MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster. Principal Findings Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results. Conclusions MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.

Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia.

Guidelines for the use of polymerase chain reaction (PCR)‐based assays to aid the diagnosis of invasive aspergillosis (IA) in high‐risk haematology patients have not been formulated. We prospectively evaluated a nested PCR assay to detect Aspergillus in blood during 95 febrile neutropenic episodes, in patients with haematological malignancy and haematopoietic stem cell transplant (HSCT) recipients. PCR results were correlated with the diagnostic classification of the 2002 European Organisation for Research and Treatment of Cancer/Mycosis Study Group. When two‐positive results were used to define an episode as ‘PCR positive’, the sensitivity, specificity, positive‐predictive value and negative predictive value for ‘proven’/‘probable’ IA (n = 13) were 100%, 75·4%, 46·4% and 100%, respectively. Consecutive positive results occurred in 61·5% of these 13 episodes. Overall, PCR positivity preceded standard diagnosis by a mean of 14 d and the median time between positive results was shorter than that in other categories of IA. All 13 episodes occurred in the setting of allogeneic HSCT recipients and acute leukaemia. If ‘eligibility’ for antifungal therapy were based on two‐positive‐PCR tests, use of empiric treatment could have been reduced by up to 37%. The nested PCR assay is a practical screening test for excluding IA. Patients with consecutive positive results or intermittent‐positive results (within 14 d) warrant immediate investigations for IA and the initiation of antifungal therapy.

Multiplex Tandem PCR: a Novel Platform for Rapid Detection and Identification of Fungal Pathogens from Blood Culture Specimens

ABSTRACT We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-α (EF1-α), and β-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans. The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (<2 h), sensitive, and specific simultaneous detection and identification of fungal pathogens directly from blood culture specimens.

Clonal Reproduction and Limited Dispersal in an Environmental Population of Cryptococcus neoformans var. gattii Isolates from Australia

ABSTRACT 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 α 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.

Clinical Utility of the Cryptococcal Antigen Lateral Flow Assay in a Diagnostic Mycology Laboratory

Background Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA) to detect cryptococcal antigen (CRAG) is reportedly more rapid and convenient than standard latex agglutination (LA), but has not yet been evaluated in a diagnostic laboratory setting. Methods One hundred and six serum, 42 cerebrospinal fluid (CSF), and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients. Results Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56) from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI) 93.6–100%) compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7–96.1%). Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56) this did not reach statistical significance (p = 0.063). Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients). Agreement between titres obtained by both methods (n = 38 samples) was imperfect; correlation between log-transformed titres (r) was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively. Conclusions Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.

Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum

Molecular typing showed genetic diversity, dismissed 2 suspected outbreaks of scedosporiosis, and identified multiple strains of the newly described species S. aurantiacum.

Nosocomial Pneumocystis jirovecii pneumonia: lessons from a cluster in kidney transplant recipients.

Background. Pneumocystis jirovecii pneumonia (PJP) is an important infection-related complication, whose mode of transmission remains uncertain. Methods. We investigated a nosocomial cluster of 14 PJP cases (11 confirmed and 3 probable) in kidney transplant recipients using epidemiological and genotyping methods. Results. Poisson regression calculated an incidence density ratio of 42.8 (95% confidence interval [CI], 14.1–129.3) versus background 0.64 cases of 1000 patient-years (P<0.001). All patients presented with respiratory failure, 10 required ventilation, two died, and six transplants failed, costing

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

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