Multicenter evaluation of a lateral-flow device test for diagnosing invasive pulmonary aspergillosis in ICU patients

Introduction: The incidence of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is increasing, and early diagnosis of the disease and treatment with antifungal drugs is critical for patient survival. Serum biomarker tests for IPA typically give false-negative results in non-neutropenic patients, and galactomannan (GM) detection, the preferred diagnostic test for IPA using bronchoalveolar lavage (BAL), is often not readily available. Novel approaches to IPA detection in ICU patients are needed. In this multicenter study, we evaluated the performance of an Aspergillus lateral-flow device (LFD) test for BAL IPA detection in critically ill patients. Methods: A total of 149 BAL samples from 133 ICU patients were included in this semiprospective study. Participating centers were the medical university hospitals of Graz, Vienna and Innsbruck in Austria and the University Hospital of Mannheim, Germany. Fungal infections were classified according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Results: Two patients (four BALs) had proven IPA, fourteen patients (sixteen BALs) had probable IPA, twenty patients (twenty-one BALs) had possible IPA and ninety-seven patients (one hundred eight BALs) did not fulfill IPA criteria. Sensitivity, specificity, negative predictive value, positive predictive value and diagnostic odds ratios for diagnosing proven and probable IPA using LFD tests of BAL were 80%, 81%, 96%, 44% and 17.6, respectively. Fungal BAL culture exhibited a sensitivity of 50% and a specificity of 85%. Conclusion: LFD tests of BAL showed promising results for IPA diagnosis in ICU patients. Furthermore, the LFD test can be performed easily and provides rapid results. Therefore, it may be a reliable alternative for IPA diagnosis in ICU patients if GM results are not rapidly available. Trial registration: ClinicalTrials.gov NCT02058316. Registered 20 January 2014

Identification of Filamentous Fungi by MALDI-TOF Mass Spectrometry: Evaluation of Three Different Sample Preparation Methods and Validation of an In-House Species Cutoff

Invasive infections caused by filamentous fungi constitute a leading cause of morbidity and mortality in immunocompromised patients. Rapid and reliable identification of filamentous fungi is essential for the early initiation of appropriate treatment. In the present study, 230 filamentous fungi isolates identified by conventional methods were investigated using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) in combination with the Filamentous Fungi Library 3.0 provided by the manufacturer. Three different sample preparation methods were applied as recommended by the manufacturer and identification rates were compared using the criteria provided by the manufacturer. Application of the more time-consuming sample preparation methods clearly improved identification at the species level. Thus, the identification rate increased from 48.9% using the simplest method to 76.1% with the most laborious procedure. Misidentifications did not occur. Furthermore, the reliability of an in-house threshold for species identification was investigated. The reduced threshold increased the rate of isolates correctly identified at the species level by up to 86.4%. As no misidentification was made at the genus level and only one misidentification of minor significance occurred at the species level, this threshold could be validated for routine use in our laboratory. In conclusion, regarding the high identification rates achieved, this commercial platform proved suitable for implementation in routine diagnosis

A Retrospective Assessment of Four Antigen Assays for the Detection of Invasive Candidiasis Among High-Risk Hospitalized Patients

Because of their high mortality rates and non-specific symptoms, invasive Candida infections pose a huge diagnostic and therapeutic challenge. In this study, we evaluated the three mannan antigen assays Platelia, Platelia Plus and Serion, and the (1-3)--d-glucan assay Fungitell in a group of high-risk (hematological and surgical) patients. Test results of 305 patients hospitalized at the Vienna General Hospital and the University Hospital of Innsbruck were retrospectively analyzed. We assessed the test accuracy by means of descriptive statistics. Nine (2.95%) patients were affected by invasive candidiasis (IC), and 25 (8.2%) patients had a probable/possible infection. The majority of patients (271; 88.9%) showed no signs of infection. The Platelia and Serion mannan assays had a low sensitivity (65% and 52%, respectively), but high specificity (98% for both tests). The newer version of the Platelia assay, the Platelia Plus, had a higher sensitivity (85%) but a lower specificity (89%). The sensitivity of the Fungitell assay was high (100%), while its specificity was low (58%). The positive predictive values were 0.48 for the Platelia and 0.41 for the Serion assay, 0.26 for the Platelia Plus and 0.09 for the Fungitell assay. Our limited, retrospective study suggests the efficacy of mannan assays as screening (Platelia Plus) and confirmatory (Serion) tests, while the Fungitell assay can be used to exclude invasive Candida infections.(VLID)362104

Development of Novel Real-Time PCR Assays for Detection and Differentiation of Eleven Medically Important Aspergillus and Candida Species in Clinical Specimens

In the present study, novel real-time PCR assays targeting the fungal ITS2 region were developed for the detection and differentiation of medically important Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) and Candida species (Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) using a LightCycler instrument. The combination of a group-specific and a universal primer with five Aspergillus or six Candida species-specific biprobes in one reaction mixture facilitated rapid screening and species differentiation by the characteristic peak melting temperatures of the biprobes. Both assays can be performed either as single assays or simultaneously in the same LightCycler run. The analytical sensitivity using pure cultures and EDTA-anticoagulated blood, cerebrospinal fluid (CSF), and tissue samples spiked with A. fumigatus and C. albicans cell suspensions was shown to be at least 1 CFU per PCR, corresponding to 5 to 10 CFU/ml blood and 10 CFU/200 μl CSF or 0.02 g tissue. To assess the clinical applicability, 26 respiratory samples, 4 tissue samples from the maxillary sinus, and 1 blood sample were retrospectively tested and real-time PCR results were compared with results from culture, histology, or a galactomannan enzyme-linked immunosorbent assay (ELISA). Twenty samples (64.5%) were both culture positive and positive by real-time PCR. Six samples (19.4%) showed no growth of fungi but were positive by real-time PCR. However, all of the tissue samples were positive by both PCR and histology. The blood sample showed no growth of Aspergillus, but aspergillosis was confirmed by positive galactomannan ELISA, histology, and PCR results. The remaining samples (16.1%) were culture and PCR negative; also, no other signs indicating fungal infection were observed. Our data suggest that the Aspergillus and Candida assays may be appropriate for use in clinical laboratories as simple and rapid screening tests for the most frequently encountered Aspergillus and Candida species and might become an important tool in the early diagnosis of fungal infections in the future

Substantial diagnostic impact of blood culture independent molecular methods in bloodstream infections : Superior performance of PCR/ESI-MS

This study analyzed the performance of different molecular technologies along with blood culture (BC) in the diagnosis of bloodstream infections (BSI) in patients from internal medicine wards - including intensive care units (ICUs) - and the emergency room. Patients with systemic inflammatory response syndrome were prospectively included. BCs and EDTA whole blood were obtained simultaneously. The latter was analyzed by PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS; IRIDICA BAC BSI assay, Abbott) and by SeptiFast (Roche). Cases were classified as BSI according to adapted European Centre for Disease Prevention and Control criteria. Out of 462 analyzed episodes, 193 with valid test results fulfilled the inclusion criteria and were further evaluated. Sixty-nine (35.8%) were classified as BSI. PCR/ESI-MS showed a significantly better overall performance than BC (p=0.004) or SeptiFast (p=0.034). Only in patients from the ICU the performance of SeptiFast was comparable to that of PCR/ESI-MS. Mainly due to the negative effect of antimicrobial pre-treatment on BC results, the cumulative performance of each of the molecular tests with BC was significantly higher than that of BC alone (p<0.001). SeptiFast and in particular the broad-range pathogen detection system PCR/ESI-MS proved to be an essential addition to BC-based diagnostics in BSI.(VLID)472612

Detection and Identification of Fungi from Fungus Balls of the Maxillary Sinus by Molecular Techniques

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found

Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

Introduction/objectivesAn increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting.MethodsForty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced.ResultsFifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2.ConclusionThis study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms

Candida auris in Austria&mdash;What Is New and What Is Different

Candida auris is a novel and emerging pathogenic yeast which represents a serious global health threat. Since its first description in Japan 2009, it has been associated with large hospital outbreaks all over the world and is often resistant to more than one antifungal drug class. To date, five C. auris isolates have been detected in Austria. Morphological characterization and antifungal susceptibility profiles against echinocandins, azoles, polyenes and pyrimidines, as well as the new antifungals ibrexafungerp and manogepix, were determined. In order to assess pathogenicity of these isolates, an infection model in Galleria mellonella was performed and whole genome sequencing (WGS) analysis was conducted to determine the phylogeographic origin. We could characterize four isolates as South Asian clade I and one isolate as African clade III. All of them had elevated minimal inhibitory concentrations to at least two different antifungal classes. The new antifungal manogepix showed high in vitro efficacy against all five C. auris isolates. One isolate, belonging to the African clade III, showed an aggregating phenotype, while the other isolates belonging to South Asian clade I were non-aggregating. In the Galleria mellonella infection model, the isolate belonging to African clade III exhibited the lowest in vivo pathogenicity. As the occurrence of C. auris increases globally, it is important to raise awareness to prevent transmission and hospital outbreaks