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Introduction: Fungal PCR has undergone considerable standardization and together with the availability of commercial assays, external quality assessment schemes and extensive performance validation data, is ready for widespread use for the screening and diagnosis of invasive fungal disease (IFD).Areas Covered: Drawing on the experience and knowledge of the leads of the various working parties of the Fungal PCR initiative, this review will address general considerations concerning the use of molecular tests for the diagnosis of IFD, before focussing specifically on the technical and clinical aspects of molecular testing for the main causes of IFD and recent technological developments.Expert Opinion: For infections caused by Aspergillus, Candida and Pneumocystis jirovecii, PCR testing is recommended, combination with serological testing will likely enhance the diagnosis of these diseases. For other IFD (e.g. Mucormycosis) molecular diagnostics, represent the only non-classical mycological approach towards diagnoses and continued performance validation and standardization has improved confidence in such testing. The emergence of antifungal resistance can be diagnosed, in part, through molecular testing. Next-generation sequencing has the potential to significantly improve our understanding of fungal phylogeny, epidemiology, pathogenesis, mycobiome/microbiome and interactions with the host, while identifying novel and existing mechanisms of antifungal resistance and novel diagnostic/therapeutic targets.

This review examined the literature on the diagnosis of invasive fungal disease (IFD) in patients undergoing solid organ transplants (SOT) to describe the diagnostic options available for this cohort. The tools available for the diagnosis of IFD in SOT patients are similar to those for patients undergoing stem cell transplants. These include (1) direct visualisation by radiography or histopathology, (2) antigenic tests using ELISA or lateral flow devices for fungal antigens, and (3) PCR-based assays that are commercially available for the two primary IFD affecting SOT patients, aspergillosis and candidiasis. Testing recipients and donors for IFD susceptibility may lead to improved prediction of IFD in SOT. The organ being transplanted has a strong bearing on the risk of IFD and the fungi that will cause disease. No single methodology can yield a definitive diagnosis so combinations of diagnostic tests targeted to the specific patient can indicate the probability of IFD.

This review examines how next-generation nucleic acid sequencing (NGS) is being used in the diagnosis of invasive fungal disease (IFD) and how well its implementation compares to PCR-based diagnosis. The comparison allows for the assessment of the advantages and potential limitations of NGS in the diagnosis of IFD. NGS is established as a method for sequencing of microbial genomes. It is increasingly being tested as a direct diagnostic method from various sample types including samples such as plasma, formalin fixed and paraffin embedded specimens, and bronchoalveolar lavage. In these cases, the methodology was generally able to identify the causative agents of IFD. NGS-based methods will play an increasingly important role in diagnosis of IFD. The major limitation is currently cost, the need to standardise methods of nucleic acid isolation, and sequence analysis to enable broad uptake and application of the method.

Invasive aspergillosis (IA) is a life-threatening complication among allogeneic hematopoietic stem cell transplant (alloSCT) recipients. Despite well known risk factors and different available assays, diagnosis of invasive aspergillosis remains challenging. 103 clinical variables from patients with hematological malignancies and subsequent alloSCT were collected. Associations between collected variables and patients with (n = 36) and without IA (n = 36) were investigated by applying univariate and multivariable logistic regression. The predictive power of the final model was tested in an independent patient cohort (23 IA cases and 25 control patients). Findings were investigated further by in vitro studies, which analysed the effect of etanercept on A. fumigatus-stimulated macrophages at the gene expression and cytokine secretion. Additionally, the release of C-X-C motif chemokine ligand 10 (CXCL10) in patient sera was studied. Low monocyte concentration (p = 4.8 × 10−06), severe GvHD of the gut (grade 2–4) (p = 1.08 × 10−02) and etanercept treatment of GvHD (p = 3.5 × 10−03) were significantly associated with IA. Our studies showed that etanercept lowers CXCL10 concentrations in vitro and ex vivo and down-regulates genes involved in immune responses and TNF-alpha signaling. Our study offers clinicians new information regarding risk factors for IA including low monocyte counts and administration of etanercept. After necessary validation, such information may be used for decision making regarding antifungal prophylaxis or closely monitoring patients at risk.

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Interactions between fungal pathogens such as Aspergillus fumigatus with host alveolar epithelium and innate immune cells are crucial in the defense against opportunistic fungal infections. In this study a simplified Transwell

Objective Recent studies into the antifungal activity of NK-cells against the Aspergillus fumigatus have presented differing accounts on their mode of antifungal activity. One of these mechanisms proposed that NK-cells may kill the fungus via the direct effects of exposure to Interferon gamma (IFN-γ). Results In this study we investigated the direct antifungal effects of recombinant human IFN-γ against a range of pathogenic fungi by measuring cellular damage using an XTT-based assay and cell viability through plate counts. It was found that 32 pg/ml of IFN-γ exhibited a significant but small antifungal effect on A. fumigatus (p = 0.02), Aspergillus flavus (p = 0.04) and Saccharomyces cerevisiae (p = 0.03), inhibiting growth by 6, 11 and 17% respectively. No significant inhibitory effects were observed in Candida species (p > 0.05 for all species tested) or Cryptococus neoformans (p = 0.98). Short term exposure (3 h) to a combination of amphotericin B (1 µg/ml) and IFN-γ (32 pg/ml) increased the effectiveness of amphotericin B against A. fumigatus and S. cerevisiae but not Candida albicans. These data suggest that IFN-γ does not possess strong antifungal activity but can enhance the effect of amphotericin B under some testing conditions against Aspergillus species.

Contains fulltext : 175113.pdf (Publisher’s version ) (Closed access) A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

In immunocompromised patients, invasive aspergillosis (IA) is the most frequent disease caused by the pathogenic mould Aspergillus fumigatus. Fever is one of the most common yet nonspecific clinical symptoms of IA. To evaluate the role of hyperthermia in the innate immune response to A. fumigatus in vitro, human monocyte-derived dendritic cells (DCs) were stimulated with germ tubes of A. fumigatus or the fungal cell wall component zymosan at 37°C or 40°C, followed by characterization of specific DC functions. While maturation of DCs was enhanced and DC phagocytic capacity was reduced at 40°C, we observed that DC viability and cytokine release were unaffected. Thus, our results suggest that hyperthermia has substantial impacts on DC function in vitro, which might also influence the course and outcome of IA in immunocompromised patients.

The central nervous system (CNS) is the most common site of dissemination during Aspergillus infection. PCR has the potential to facilitate early diagnosis of CNS aspergillosis, which could assist in reducing disease mortality. In two experiments, neutropenic CD-1 male mice were infected intracranially with 5×106 conidia of Aspergillus fumigatus. At time points up to 120 h after infection, mice were euthanized and samples of blood, brain, spinal cord and cerebrospinal fluid (CSF) were taken. The brain fungal burden was determined by quantitative culture, and fungal DNA was detected by quantitative PCR. Plating for A. fumigatus from the brain confirmed that all mice had burdens of log10 >3 from 4 to 120 h after infection. A. fumigatus DNA was detected in blood (88 %), brain (96 %), CSF (52 %) and spinal cord (92 %) samples. The brain and spinal cord contained the highest concentrations of fungal DNA. Adapting the extraction protocol to maximize yield from small sample volumes (10 µl CSF or 200 µl blood) allowed PCR detection of A. fumigatus in infected mice, suggesting the use of CSF and blood as diagnostic clinical samples for CNS aspergillosis.

Pochonia chlamydosporia var. catenulata is a potential biocontrol agent against root-knot nematodes. Diagnosis of isolates has relied on morphological identification, and is both time-consuming and difficult. beta-tubulin primers have been developed for the identification of this fungus that were specific enough to distinguish between varieties of the fungus within the same species. Separate primers have been developed for the specific detection of P. chlamydosporia var. catenulata based on ITS sequences, which were able to detect the fungus in soil from various sites in Cuba where the biocontrol agent had been added. When the PCR diagnosis was combined with serial dilution of soil samples on selective medium, colonies were rapidly identified. The fungus was still present, albeit at low densities, in soils inoculated five years previously. The development of a baiting method allowed quick in situ screening of the isolates' ability to infect nematode eggs, and when combined with PCR diagnosis both varieties of the fungus could be detected in infected eggs. RFLP analysis of ITS sequences from P. chlamydosporia provided an extra level of discrimination between isolates.

Fungal infection of nails, onychomycosis, is predominantly caused by Trichophyton rubrum. This infection is an important public health concern due to its persistent nature and high recurrence rates. Alternative treatments are urgently required. One such alternative is phototherapy involving the action of photothermal or photochemical processes. The aim of this novel study was to assess which wavelengths within the ultraviolet (UV) spectrum were inhibitory and equally important nail transmissible. Initial irradiations of T. rubrum spore suspensions were carried out using a tunable wavelength lamp system (fluence ≤3.1 J/cm(2)) at wavelengths between 280 and 400 nm (UVC to UVA) to evaluate which wavelengths prevented fungal growth. Light-emitting diodes (LEDs) of defined wavelengths were subsequently chosen with a view to evaluate and potentially implement this technology as a low-cost "in-home" treatment. Our experiments demonstrated that exposure at 280 nm using an LED with a fluence as low as 0.5 J/cm(2) was inhibitory, i.e., no growth following a 2-week incubation (p0.05; one-way ANOVA), while exposure to longer wavelengths was not. A key requirement for the use of phototherapy in the treatment of onychomycosis is that it must be nail transmissible. Our results indicate that the treatment with UVC is not feasible given that there is no overlap between the antifungal activity observed at 280 nm and transmission through the nail plate. However, a potential indirect application of this technology could be the decontamination of reservoirs of infection such as the shoes of infected individuals, thus preventing reinfection.

Despite the strong interest in the NK cell-mediated immunity toward malignant cells and viruses, there is a relative lack of data on the interplay between NK cells and filamentous fungi, especially Aspergillus fumigatus, which is the major cause of invasive aspergillosis. By studying the in vitro interaction between human NK cells and A. fumigatus, we found only germinated morphologies to be highly immunogenic, able to induce a Th1-like response, and capable of upregulating cytokines such as IFN-γ and TNF-α. Moreover, priming NK cells with human rIL-2 and stimulating NK cells by direct NK cell–pathogen contact were essential to induce damage against A. fumigatus. However, the most interesting finding was that NK cells did not mediate anti-Aspergillus cytotoxicity through degranulation of their cytotoxic proteins (perforin, granzymes, granulysine), but via an alternative mechanism involving soluble factor(s). To our knowledge, our study is the first to demonstrate that IFN-γ, released by NK cells, directly damages A. fumigatus, attributing new properties to both human NK cells and IFN-γ and suggesting them as possible therapeutic tools against IA.

Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an α-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae . Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.

The dermaseptins are a family of antimicrobial peptides from the tree-frog Phyllomedusa sauvagii. Yeast exposed to dermaseptin S3(1-16), a truncated derivative of dermaseptin S3 with full activity, showed diagnostic markers of yeast apoptosis: the appearance of reactive oxygen species and fragmentation of nuclear DNA. This process was independent of the yeast caspase, Yca1p. Screening of a non-essential gene deletion collection in yeast identified genes that conferred resistance to dermaseptin S3(1-16): izh2Delta, izh3Delta, stm1Delta and aif1Delta, all known to be involved in regulating yeast apoptosis. The appearance of apoptotic markers was reduced in these strains when exposed to the peptide. Dermaseptin S3(1-16) was shown to interact with DNA, and cause DNA damage in vivo, a process known to trigger apoptosis. Supporting this, a dermaseptin S3(1-16) affinity column specifically purified Stm1p, Mre11p and Htb2p; DNA-binding proteins implicated in yeast apoptosis and DNA repair. Thus, amphibians may have evolved a mechanism to induce cell suicide in invading fungal pathogens.

The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.

The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root knot and cyst nematodes. It produces an alkaline serine protease, VCP1, during infection of nematode eggs. The gene encoding VCP1 was sequenced and the sequences of cDNAs from six isolates from different nematode hosts were compared. The gene encoding VCP1 was similar to PR1 from Metarhizium anisopliae with similar regulatory elements. Comparison of translated cDNA sequences revealed two amino acid polymorphisms at positions 65 and 99, indicating a difference between isolates from cyst and root nematodes. The positions and nature of the polymorphisms indicated that the two forms of VCP1 might have different properties and this was tested with five chromogenic polypeptide substrates. Enzyme assays revealed the two forms differed in their abilities to utilise Succ-Ala-Ala-Pro-Phe-pNa and Succ-Ala-Val-Pro-Phe-pNa, suggesting different amino acid affinities at the S3 binding region. This indicates host related genetic variation in VCP1 between isolates of P. chlamydosporia isolated from different nematode hosts, which might contribute to host preference. Such differences may be important in future exploitation of P. chlamydosporia as a nematode biocontrol agent.