Detection of the 'Big Five' mold killers of humans: Aspergillus, Fusarium, Lomentospora, Scedosporium and Mucormycetes.

Fungi are an important but frequently overlooked cause of morbidity and mortality in humans. Life-threatening fungal infections mainly occur in immunocompromised patients, and are typically caused by environmental opportunists that take advantage of a weakened immune system. The filamentous fungus Aspergillus fumigatus is the most important and well-documented mold pathogen of humans, causing a number of complex respiratory diseases, including invasive pulmonary aspergillosis, an often fatal disease in patients with acute leukemia or in immunosuppressed bone marrow or solid organ transplant recipients. However, non-Aspergillus molds are increasingly reported as agents of disseminated diseases, with Fusarium, Scedosporium, Lomentospora and mucormycete species now firmly established as pathogens of immunosuppressed and immunocompetent individuals. Despite well-documented risk factors for invasive fungal diseases, and increased awareness of the risk factors for life-threatening infections, the number of deaths attributable to molds is likely to be severely underestimated driven, to a large extent, by the lack of readily accessible, cheap, and accurate tests that allow detection and differentiation of infecting species. Early diagnosis is critical to patient survival but, unlike Aspergillus diseases, where a number of CE-marked or FDA-approved biomarker tests are now available for clinical diagnosis, similar tests for fusariosis, scedosporiosis and mucormycosis remain experimental, with detection reliant on insensitive and slow culture of pathogens from invasive bronchoalveolar lavage fluid, tissue biopsy, or from blood. This review examines the ecology, epidemiology, and contemporary methods of detection of these mold pathogens, and the obstacles to diagnostic test development and translation of novel biomarkers to the clinical setting.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

Detection of human pathogenic Fusarium species in hospital and communal sink biofilms by using a highly specific monoclonal antibody.

The fungus Fusarium is well known as a plant pathogen, but has recently emerged as an opportunistic pathogen of humans. Habitats providing direct human exposure to infectious propagules are largely unknown, but there is growing evidence that plumbing systems are sources of human pathogenic strains in the Fusarium solani species complex (FSSC) and Fusarium oxysporum species complex (FOSC), the most common groups infecting humans. Here, a newly developed Fusarium-specific monoclonal antibody (mAb ED7) was used to track FSSC and FOSC strains in sink drain biofilms by detecting its target antigen, an extracellular 200 kDa carbohydrate, in saline swabs. The antigen was detectable in 52% of swab samples collected from sinks across a University campus and a tertiary care hospital. The mAb was 100% accurate in detecting FSSC, FOSC, and F. dimerum species complex (FDSC) strains that were present, as mixed fungal communities, in 83% of sink drain biofilms. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of culturable yeasts and molds that were recovered using mycological culture, while translation elongation factor (TEF)-1α analysis of Fusarium isolates included FSSC 1-a, FOSC 33, and FDSC ET-gr, the most common clinical pathotypes in each group.

Identifying risk factors for exposure to culturable allergenic moulds in energy efficient homes by using highly specific monoclonal antibodies.

The aim of this study was to determine the accuracy of monoclonal antibodies (mAbs) in identifying culturable allergenic fungi present in visible mould growth in energy efficient homes, and to identify risk factors for exposure to these known allergenic fungi. Swabs were taken from fungal contaminated surfaces and culturable yeasts and moulds isolated by using mycological culture. Soluble antigens from cultures were tested by ELISA using mAbs specific to the culturable allergenic fungi Aspergillus and Penicillium spp., Ulocladium, Alternaria, and Epicoccum spp., Cladosporium spp., Fusarium spp., and Trichoderma spp. Diagnostic accuracies of the ELISA tests were determined by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2-encoding regions of recovered fungi following ELISA. There was 100% concordance between the two methods, with ELISAs providing genus-level identity and ITS sequencing providing species-level identities (210 out of 210 tested). Species of Aspergillus/Penicillium, Cladosporium, Ulocladium/Alternaria/Epicoccum, Fusarium and Trichoderma were detected in 82% of the samples. The presence of condensation was associated with an increased risk of surfaces being contaminated by Aspergillus/Penicillium spp. and Cladosporium spp., whereas moisture within the building fabric (water ingress/rising damp) was only associated with increased risk of Aspergillus/Penicillium spp. Property type and energy efficiency levels were found to moderate the risk of indoor surfaces becoming contaminated with Aspergillus/Penicillium and Cladosporium which in turn was modified by the presence of condensation, water ingress and rising damp, consistent with previous literature.

NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus.

The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2-NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics.

Indoor fungal diversity and asthma: a meta-analysis and systematic review of risk factors.

BACKGROUND: Indoor dampness increases the risk of indoor fungal growth. A complex interaction between occupant behaviors and the built environment are thought to affect indoor fungal concentrations and species diversity, which are believed to increase the risk of having asthma, exacerbation of asthma symptoms, or both. To date, no systematic review has investigated this relationship. OBJECTIVE: This review aims to assess the relationship between exposure to indoor fungi identified to the genera or species level on asthma outcomes in children and adults. METHODS: Ten databases were systematically searched on April 18, 2013, and limited to articles published since 1990. Reference lists were independently screened by 2 reviewers, and authors were contacted to identify relevant articles. Data were extracted from included studies meeting our eligibility criteria by 2 reviewers and quality assessed by using the Newcastle-Ottawa scale designed for assessment of case-control and cohort studies. RESULTS: Cladosporium, Alternaria, Aspergillus, and Penicillium species were found to be present in higher concentrations in homes of asthmatic participants. Exposure to Penicillium, Aspergillus, and Cladosporium species were found to be associated with increased risk of reporting asthma symptoms by a limited number of studies. The presence of Cladosporium, Alternaria, Aspergillus, and Penicillium species increased the exacerbation of current asthma symptoms by 36% to 48% compared with those exposed to lower concentrations of these fungi, as shown by using random-effect estimates. Studies were of medium quality and showed medium-high heterogeneity, but evidence concerning the specific role of fungal species was limited. CONCLUSION: Longitudinal studies assessing increased exposure to indoor fungi before the development of asthma symptoms suggests that Penicillium, Aspergillus, and Cladosporium species pose a respiratory health risk in susceptible populations. Increased exacerbation of current asthma symptoms in children and adults were associated with increased levels of Penicillium, Aspergillus, Cladosporium, and Alternaria species, although further work should consider the role of fungal diversity and increased exposure to other fungal species.

Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival.

The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Taken together, these results show that while melanin plays a protective role in the survival of the pathogen to oxidative killing and UV radiation, melanin does not contribute to its resistance to amphotericin B.

Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds.

Immunodetection of fungal and oomycete pathogens: established and emerging threats to human health, animal welfare and global food security.

Filamentous fungi (moulds), yeast-like fungi, and oomycetes cause life-threatening infections of humans and animals and are a major constraint to global food security, constituting a significant economic burden to both agriculture and medicine. As well as causing localized or systemic infections, certain species are potent producers of allergens and toxins that exacerbate respiratory diseases or cause cancer and organ damage. We review the pathogenic and toxigenic organisms that are etiologic agents of both animal and plant diseases or that have recently emerged as serious pathogens of immunocompromised individuals. The use of hybridoma and phage display technologies and their success in generating monoclonal antibodies for the detection and control of fungal and oomycete pathogens are explored. Monoclonal antibodies hold enormous potential for the development of rapid and specific tests for the diagnosis of human mycoses, however, unlike plant pathology, their use in medical mycology remains to be fully exploited.

Indoor Fungal Exposure and Allergic Respiratory Disease.

A gathering body of evidence has repeatedly revealed associations between indoor fungi and initiation, promotion, and exacerbation of allergic respiratory disease. The relationship between the exposure and outcome are complicated by the difficulties in measuring both exposure and outcome, the multifactorial nature of the disease, and the wide range of potential confounders. New technologies are becoming available that may enable better measurement of exposure and tighter case definitions so as to build more confidence in the associations discovered. The growing strength of the evidence base will aid the design of future public health interventions and generate new hypotheses on the cause of the rapid increase in allergic respiratory disease prevalence.

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.

Novel tests for diagnosis of invasive aspergillosis in patients with underlying respiratory diseases.

RATIONALE: Invasive pulmonary aspergillosis has been increasingly reported in nonneutropenic patients, including those with underlying respiratory diseases. OBJECTIVES: We compared the diagnostic performances of galactomannan, 1,3-ß-D-glucan, and Aspergillus-specific lateral-flow device tests with that of conventional culture by using bronchoalveolar lavage fluid samples from patients with underlying respiratory diseases. METHODS: We analyzed 268 bronchoalveolar lavage samples from 221 patients with underlying respiratory diseases (and without hematologic malignancy or previous solid organ transplantation) that were collected for routine microbiological workup between February 2012 and May 2014 at the University Hospital of Graz, Austria. Invasive pulmonary aspergillosis was defined according to European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria modified for patients with respiratory diseases. MEASUREMENTS AND MAIN RESULTS: Thirty-one patients (14%) had probable or proven, 25 possible, and the remaining 165 patients no invasive pulmonary aspergillosis. Probable/proven aspergillosis was associated with a significantly higher (P = 0.034) 30-day mortality rate of 32%. Sensitivities, specificities, and diagnostic odd ratios differed markedly between galactomannan (cut-off 0.5: optical density index, 0.97, 0.81, 124.4; cut-off 1.0: 0.97, 0.93, 422.1; cut-off 3.0: 0.61, 0.99, 109.8), ß-D-glucan (cut-off 80 pg/ml: 0.90, 0.42, 6.57; cut-off 200 pg/ml: 0.70, 0.61, 3.7), lateral-flow device tests (0.77, 0.92, 41.8), and mycological culture (0.29, 0.97, 14). CONCLUSIONS: Probable or proven invasive pulmonary aspergillosis was diagnosed in 14% of our study population and associated with significantly higher 30-day mortality rates. Although the performance of ß-D-glucan was limited by low specificity and that of mycological culture by low sensitivity, the Aspergillus lateral-flow device seems to be a promising alternative to galactomannan testing, which remains the diagnostic gold standard for aspergillosis. Clinical trial registered with www.clinicaltrials.gov (NCT 02058316).

Performance of lateral flow device and galactomannan for the detection of Aspergillus species in bronchoalveolar fluid of patients at risk for invasive pulmonary aspergillosis.

Early diagnosis of invasive pulmonary aspergillosis (IPA) remains difficult due to the variable performance of the tests used. We compared the performance characteristics of Aspergillus lateral flow device (LFD) in bronchoalveolar lavage (BAL) vs. BAL-galactomannan (GM), for the diagnosis of IPA. 311 BAL specimens were prospectively collected from patients who underwent bronchoscopy from January to May 2013. Patients at risk for IPA were divided into haematological malignancy (HEM) and non-HEM groups: solid organ transplants (SOT) (lung transplant (LT) and non-LT SOT); chronic steroid use (CSU); solid tumour (STU) and others. We identified 96 patients at risk for IPA; 89 patients (93%) were in the non-HEM groups: SOT 57 (LT, 46, non-LT SOT, 11); CSU 21; STU 6, other 5. Only three patients met criteria for IA (two probable; one possible). Overall sensitivity (SS) was 66% for both and specificity (SP) was 94% vs. 52% for LFD and GM respectively. LFD and GM performance was similar in the HEM group (SS 100% for both and SP 83% vs. 100% respectively). LFD performance was better than GM among non-HEM SOT patients (P = 0.02). Most false-positive GM results occurred in the SOT group (50.8%), especially among LT patients (56.5%). LFD performance was superior with an overall SP of 95.6% in SOT (P < 0.002) and 97% in LT patients (P = 0.0008). LFD is a rapid and simple test that can be performed on BAL to rule out IPA.

Diagnostic accuracy of the Aspergillus-specific bronchoalveolar lavage lateral-flow assay in haematological malignancy patients.

We evaluated the performance of the Aspergillus-specific lateral-flow device (LFD) test for diagnosing invasive pulmonary aspergillosis (IPA) in patients with underlying haematological malignancies. Participating centres were the two Austrian University Hospitals of Graz and Innsbruck. LFD performance was evaluated with 95 bronchoalveolar lavage fluid (BALF) samples from 72 patients collected prospectively in Graz, and with 24 BALF bio bank samples from 23 patients (21 samples with probable IPA) in Innsbruck. Invasive fungal infections were classified according to the revised European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria. Overall, 27 patients (30 samples) had probable IPA, 32 (43 samples) possible and 36 (46 samples) did not fulfil IPA criteria. The vast majority of patients - in particular those with probable IPA - received mould-active treatment before bronchoscopy. Sensitivity, specificity, positive predictive value and negative-predictive-value for probable IPA diagnosis using the BALF-LFD test were 71%, 76%, 35% and 94% for the Graz cohort. Sensitivity of the BALF-LFD test for probable IPA was 57% in Innsbruck bio bank samples. Our results indicate that the BALF-LFD-test provides fast results with moderate sensitivities in patients with underlying haematological malignancies. Similar to other diagnostic tests and biomarkers sensitivity of the test may be influenced by ongoing systemic mould-active treatment.

The potential for rapid antigen testing for mucormycosis in the context of COVID-19.

INTRODUCTION: Mucormycosis is a highly aggressive angio-invasive disease of humans caused by Mucorales fungi. Prior to the COVID-19 pandemic, mucormycosis was a rare mycosis typically seen in immunocompromised patients with hematological malignancies or in transplant recipients. During the second wave of the pandemic, there was a dramatic increase in the disease, especially in India where a unique set of circumstances led to large numbers of life-threatening and disfiguring rhino-orbital-cerebral mucormycosis (ROCM) infections. AREAS COVERED: The review examines mucormycosis as a super-infection of COVID-19 patients, and the risk factors for COVID-19-associated mucormycosis (CAM) that drove the ROCM epidemic in India. The limitations of current diagnostic procedures are identified, and the measures needed to improve the speed and accuracy of detection discussed. EXPERT OPINION: Despite increased awareness, global healthcare systems remain unprepared for further outbreaks of ROCM. Current diagnosis of the disease is slow and inaccurate, negatively impacting on patient survival. This is most evident in low- to middle-income countries which lack suitably equipped diagnostic facilities for rapid identification of the infecting pathogens. Rapid antigen testing using point-of-care lateral-flow assays could potentially have aided in the quick and accurate diagnosis of the disease, allowing earlier intervention with surgery and Mucorales-active antifungal drugs.

A Lateral-Flow Device for the Rapid Detection of Scedosporium Species.

Scedosporium species are human pathogenic fungi, responsible for chronic, localised, and life-threatening disseminated infections in both immunocompetent and immunocompromised individuals. The diagnosis of Scedosporium infections currently relies on non-specific CT, lengthy and insensitive culture from invasive biopsy, and the time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests that detect Scedosporium-specific biomarkers. Here, we report the development of a rapid (30 min) and sensitive (pmol/L sensitivity) lateral-flow device (LFD) test, incorporating a Scedosporium-specific IgG1 monoclonal antibody (mAb), HG12, which binds to extracellular polysaccharide (EPS) antigens between ~15 kDa and 250 kDa secreted during the hyphal growth of the pathogens. The test is compatible with human serum and allows for the detection of the Scedosporium species most frequently reported as agents of human disease (Scedosporium apiospermum, Scedosporium aurantiacum, and Scedosporium boydii), with limits of detection (LODs) of the EPS biomarkers in human serum of ~0.81 ng/mL (S. apiospermum), ~0.94 ng/mL (S. aurantiacum), and ~1.95 ng/mL (S. boydii). The Scedosporium-specific LFD (ScedLFD) test therefore provides a potential novel opportunity for the detection of infections caused by different Scedosporium species.

The pathobiology of human fungal infections.

Human fungal infections are a historically neglected area of disease research, yet they cause more than 1.5 million deaths every year. Our understanding of the pathophysiology of these infections has increased considerably over the past decade, through major insights into both the host and pathogen factors that contribute to the phenotype and severity of these diseases. Recent studies are revealing multiple mechanisms by which fungi modify and manipulate the host, escape immune surveillance and generate complex comorbidities. Although the emergence of fungal strains that are less susceptible to antifungal drugs or that rapidly evolve drug resistance is posing new threats, greater understanding of immune mechanisms and host susceptibility factors is beginning to offer novel immunotherapeutic options for the future. In this Review, we provide a broad and comprehensive overview of the pathobiology of human fungal infections, focusing specifically on pathogens that can cause invasive life-threatening infections, highlighting recent discoveries from the pathogen, host and clinical perspectives. We conclude by discussing key future challenges including antifungal drug resistance, the emergence of new pathogens and new developments in modern medicine that are promoting susceptibility to infection.

Interlaboratory and interstudy reproducibility of a novel lateral-flow device and influence of antifungal therapy on detection of invasive pulmonary aspergillosis.

Interest in lateral-flow devices (LFDs) as potential point-of-care assays for the diagnosis of infectious diseases has increased. Our objective was to evaluate the interlaboratory and interstudy reproducibility and the effects of antifungal therapy on an LFD developed for invasive pulmonary aspergillosis (IPA) detection. An established neutropenic guinea pig model of IPA caused by Aspergillus fumigatus was used. At predetermined time points (1 h and 3, 5, and 7 days postinoculation), blood and bronchoalveolar lavage (BAL) fluid were collected from infected and uninfected animals. In a separate experiment, guinea pigs were treated with posaconazole (10 mg/kg of body weight orally [p.o.] twice a day [BID]), voriconazole (10 mg/kg p.o. BID), liposomal amphotericin B (10 mg/kg intraperitoneally [i.p.] once a day [QD]), or caspofungin (2 mg/kg i.p. QD), and samples were collected on days 7 and 11. Each laboratory independently evaluated the IgG monoclonal antibody-based LFD. Galactomannan and (1 → 3)-ß-D-glucan were also measured using commercially available kits. Good interlaboratory agreement was observed with the LFD, as the results for 97% (32/33) of the serum and 78.8% (26/33) of the BAL fluid samples from infected animals were in agreement. Good interstudy agreement was also observed. The serum sensitivity of each surrogate-marker assay was reduced in animals treated with antifungals. In contrast, these markers remained elevated within the BAL fluids of treated animals, which was consistent with the fungal burden and histopathology results. These results demonstrate that the LFD assay is reproducible between different laboratories and studies. However, the sensitivity of this assay and other markers of IPA may be reduced with serum in the presence of antifungal therapy.

Development of a monoclonal antibody and a lateral-flow device for the rapid detection of a Mucorales-specific biomarker.

Mucoromycosis is a highly aggressive angio-invasive disease of humans caused by fungi in the zygomycete order, Mucorales. While Rhizopus arrhizus is the principal agent of mucoromycosis, other Mucorales fungi including Apophysomyces, Cunninghamella, Lichtheimia, Mucor, Rhizomucor and Syncephalastrum are able to cause life-threatening rhino-orbital-cerebral, pulmonary, gastro-intestinal and necrotising cutaneous infections in humans. Diagnosis of the disease currently relies on non-specific CT, lengthy and insensitive culture from invasive biopsy, and time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests that detect Mucorales-specific biomarkers of infection, and which allow point-of-care diagnosis of mucoromycosis. Here, we report the development of an IgG2b monoclonal antibody (mAb), TG11, which binds to extracellular polysaccharide (EPS) antigens of between 20 kDa and 250 kDa secreted during hyphal growth of Mucorales fungi. The mAb is Mucorales-specific and does not cross-react with other yeasts and molds of clinical importance including Aspergillus, Candida, Cryptococcus, Fusarium, Lomentospora and Scedosporium species. Using the mAb, we have developed a Competitive lateral-flow device that allows rapid (30 min) detection of the EPS biomarker in human serum and bronchoalveolar lavage (BAL), with a limit of detection (LOD) in human serum of ~100 ng/mL serum (~224.7 pmol/L serum). The LFD therefore provides a potential novel opportunity for detection of mucoromycosis caused by different Mucorales species.