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 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.

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.

Development of a Monoclonal Antibody and a Serodiagnostic Lateral-Flow Device Specific to Rhizopus arrhizus (Syn. R. oryzae), the Principal Global Agent of Mucormycosis in Humans.

Mucormycosis is a highly aggressive angio-invasive disease of humans caused by fungi in the zygomycete order, Mucorales. Though a number of different species can cause mucormycosis, the principal agent of the disease worldwide is Rhizopus arrhizus, which accounts for the majority of rhino-orbital-cerebral, pulmonary, and disseminated infections in immunocompromised individuals. It is also the main cause of life-threatening infections in patients with poorly controlled diabetes mellitus, and in corticosteroid-treated patients with SARS-CoV-2 infection, where it causes the newly described disease, COVID-19-associated mucormycosis (CAM). Diagnosis currently relies on non-specific CT, a lengthy and insensitive culture from invasive biopsy, and a time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests for the disease that detect biomarkers of infection, and which allow point-of-care diagnosis. Here, we report the development of an IgG1 monoclonal antibody (mAb), KC9, which is specific to Rhizopus arrhizus var. arrhizus (syn. Rhizopus oryzae) and Rhizopus arrhizus var. delemar (Rhizopus delemar), and which binds to a 15 kDa extracellular polysaccharide (EPS) antigen secreted during hyphal growth of the pathogen. Using the mAb, we have developed a competitive lateral-flow device (LFD) that allows rapid (30 min) and sensitive (~50 ng/mL running buffer) detection of the EPS biomarker, and which is compatible with human serum (limit of detection of ~500 ng/mL) and bronchoalveolar lavage fluid (limit of detection of ~100 ng/mL). The LFD, therefore, provides a potential novel opportunity for the non-invasive detection of mucormycosis caused by Rhizopus arrhizus.

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.

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test's diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus.

Pre-clinical Imaging of Invasive Candidiasis Using ImmunoPET/MR.

The human commensal yeast Candida is the fourth most common cause of hospital-acquired bloodstream infections, with Candida albicans accounting for the majority of the >400,000 life-threatening infections annually. Diagnosis of invasive candidiasis (IC), a disease encompassing candidemia (blood-borne yeast infection) and deep-seated organ infections, is a major challenge since clinical manifestations of the disease are indistinguishable from viral, bacterial and other fungal diseases, and diagnostic tests for biomarkers in the bloodstream such as PCR, ELISA, and pan-fungal ß-D-glucan lack either standardization, sensitivity, or specificity. Blood culture remains the gold standard for diagnosis, but test sensitivity is poor and turn-around time slow. Furthermore, cultures can only be obtained when the yeast resides in the bloodstream, with samples recovered from hematogenous infections often yielding negative results. Consequently, there is a pressing need for a diagnostic test that allows the identification of metastatic foci in deep-seated Candida infections, without the need for invasive biopsy. Here, we report the development of a highly specific mouse IgG3 monoclonal antibody (MC3) that binds to a putative ß-1,2-mannan epitope present in high molecular weight mannoproteins and phospholipomannans on the surface of yeast and hyphal morphotypes of C. albicans, and its use as a [64Cu]NODAGA-labeled tracer for whole-body pre-clinical imaging of deep-seated C. albicans infections using antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI). When used in a mouse intravenous (i.v.) challenge model that faithfully mimics disseminated C. albicans infections in humans, the [64Cu]NODAGA-MC3 tracer accurately detects infections of the kidney, the principal site of blood-borne candidiasis in this model. Using a strain of the emerging human pathogen Candida auris that reacts with MC3 in vitro, but which is non-infective in i.v. challenged mice, we demonstrate the accuracy of the tracer in diagnosing invasive infections in vivo. This pre-clinical study demonstrates the principle of using antibody-guided molecular imaging for detection of deep organ infections in IC, without the need for invasive tissue biopsy.

Molecular Imaging of Invasive Pulmonary Aspergillosis Using ImmunoPET/MRI: The Future Looks Bright.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immuno-compromised humans caused by the ubiquitous environmental mold Aspergillus. Biomarker tests for the disease lack sensitivity and specificity, and culture of the fungus from invasive lung biopsy is slow, insensitive, and undesirable in critically ill patients. A computed tomogram (CT) of the chest offers a simple non-intrusive diagnostic procedure for rapid decision making, and so is used in many hematology units to drive antifungal treatment. However, radiological indicators that raise the suspicion of IPA are either transient signs in the early stages of the disease or not specific for Aspergillus infection, with other angio-invasive molds or bacterial pathogens producing comparable radiological manifestations in a chest CT. Improvements to the specificity of radiographic imaging of IPA have been attempted by coupling CT and positron emission tomography (PET) with [18F]fluorodeoxyglucose ([18F]FDG), a marker of metabolic activity well suited to cancer imaging, but with limited use in invasive fungal disease diagnostics due to its inability to differentiate between infectious etiologies, cancer, and inflammation. Bioluminescence imaging using single genetically modified strains of Aspergillus fumigatus has enabled in vivo monitoring of IPA in animal models of disease. For in vivo detection of Aspergillus lung infections in humans, radiolabeled Aspergillus-specific monoclonal antibodies, and iron siderophores, hold enormous potential for clinical diagnosis. This review examines the different experimental technologies used to image IPA, and recent advances in state-of-the-art molecular imaging of IPA using antibody-guided PET/magnetic resonance imaging (immunoPET/MRI).

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.

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant ß1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.

Transcriptional reprogramming underpins enhanced plant growth promotion by the biocontrol fungus Trichoderma hamatum GD12 during antagonistic interactions with Sclerotinia sclerotiorum in soil.

The free-living soil fungus Trichoderma hamatum strain GD12 is notable amongst Trichoderma strains in both controlling plant diseases and stimulating plant growth, a property enhanced during its antagonistic interactions with pathogens in soil. These attributes, alongside its markedly expanded genome and proteome compared with other biocontrol and plant growth-promoting Trichoderma strains, imply a rich potential for sustainable alternatives to synthetic pesticides and fertilizers for the control of plant disease and for increasing yields. The purpose of this study was to investigate the transcriptional responses of GD12 underpinning its biocontrol and plant growth promotion capabilities during antagonistic interactions with the pathogen Sclerotinia sclerotiorum in soil. Using an extensive mRNA-seq study capturing different time points during the pathogen-antagonist interaction in soil, we show that dynamic and biphasic signatures in the GD12 transcriptome underpin its biocontrol and plant (lettuce) growth-promoting activities. Functional predictions of differentially expressed genes demonstrate the enrichment of transcripts encoding proteins involved in transportation and oxidation-reduction reactions during both processes and an over-representation of siderophores. We identify a biphasic response during biocontrol characterized by a significant induction of transcripts encoding small-secreted cysteine-rich proteins, secondary metabolite-producing gene clusters and genes unique to GD12. These data support the hypothesis that Sclerotinia biocontrol is mediated by the synthesis and secretion of antifungal compounds and that GD12's unique reservoir of uncharacterized genes is actively recruited during the effective biological control of a plurivorous plant pathogen.

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.

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.

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.

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.

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.

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).

Breaking the mould - novel diagnostic and therapeutic strategies for invasive pulmonary aspergillosis in the immune deficient patient.

Invasive pulmonary aspergillosis (IPA) caused by the ubiquitous environmental fungus Aspergillus is a frequently fatal lung disease of immunocompromised humans accounting for more than 200,000 infections each year, with an associated mortality rate of 30-90%. This review addresses the current status of IPA diagnosis and treatment and the urgent need to develop accurate, non-invasive strategies for identifying pulmonary infections in the ever-expanding population of immune deficient patients at risk of acquiring opportunistic fungal infections including hematological malignancy and hematopoetic stem cell transplant patients. Recent advances in the use of an Aspergillus-specific monoclonal antibody, JF5, for point-of-care diagnosis of IPA using lateral-flow technology is examined, as is its use in PET/MRI bioimaging and radio-immunotherapy using radionuclide-labeled single chain antibody fragments, Fab fragments, and a fully humanized JF5 derivative.

Detection of invasive aspergillosis.

Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus is a frequent and life-threatening complication of chemotherapy and bone marrow transplantation with high rates of mortality and morbidity. Diagnosis of IA is complex and can only be confirmed by identification of the fungus in biopsy samples. Capturing tissue for diagnosis is in itself hazardous, and because of this many patients receive empirical antifungal treatment rather than undergo biopsy. However, the treatment carries with it significant side effects and is prohibitively expensive. Because of this, attempts have been made to develop specific and sensitive diagnostic tests that can be used to track the early onset of infection and permit rational administration of antifungal drugs. Early attempts at nonculture-based diagnosis using human immune serum to detect circulating Aspergillus antigens proved unreliable, and so focus turned to hybridoma technology and the use of monoclonal antibodies (MAbs) to detect signature molecules of infection. Detection of one such signature molecule, galactomannan (and associated galactomannoprotein molecules), forms the basis of the commercial Platelia enzyme immunoassay (EIA), an assay that has found widespread use in IA diagnosis. Nevertheless, concerns surrounding its accuracy mean that alternative strategies to diagnosis have been sought including detection of the fungal cell wall component (1-->3)-beta-d-glucan and polymerase chain reaction (PCR). The poor specificity of "panfungal" (1-->3)-beta-d-glucan tests and current lack of standardization of PCR assays have led to the recent development of next-generation MAb-based assays that detect surrogate markers of infection and that have been incorporated into "point-of-care" diagnostic devices. This chapter examines the development of antibody-antigen, (1-->3)-beta-d-glucan, and nucleic acid-based approaches to IA detection, current concerns surrounding accurate disease diagnosis, and how animal models of infection can be used to inform assay development and validation.