Diagnosis of invasive fungal infections: challenges and recent developments.

BACKGROUND: The global burden of invasive fungal infections (IFIs) has shown an upsurge in recent years due to the higher load of immunocompromised patients suffering from various diseases. The role of early and accurate diagnosis in the aggressive containment of the fungal infection at the initial stages becomes crucial thus, preventing the development of a life-threatening situation. With the changing demands of clinical mycology, the field of fungal diagnostics has evolved and come a long way from traditional methods of microscopy and culturing to more advanced non-culture-based tools. With the advent of more powerful approaches such as novel PCR assays, T2 Candida, microfluidic chip technology, next generation sequencing, new generation biosensors, nanotechnology-based tools, artificial intelligence-based models, the face of fungal diagnostics is constantly changing for the better. All these advances have been reviewed here giving the latest update to our readers in the most orderly flow. MAIN TEXT: A detailed literature survey was conducted by the team followed by data collection, pertinent data extraction, in-depth analysis, and composing the various sub-sections and the final review. The review is unique in its kind as it discusses the advances in molecular methods; advances in serology-based methods; advances in biosensor technology; and advances in machine learning-based models, all under one roof. To the best of our knowledge, there has been no review covering all of these fields (especially biosensor technology and machine learning using artificial intelligence) with relevance to invasive fungal infections. CONCLUSION: The review will undoubtedly assist in updating the scientific community's understanding of the most recent advancements that are on the horizon and that may be implemented as adjuncts to the traditional diagnostic algorithms.

Implementation of Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis in European Hospitals: A Survey from Belgium and a Literature Review of Test Performances in Different Patient Populations.

OBJECTIVES: Diagnosis of invasive aspergillosis is based on a combination of criteria, of which the detection of Aspergillus galactomannan (GM) often is decisive. To date, the most commonly used method to determine GM is an enzyme-linked immune assay (EIA). But since a few years lateral flow assays (LFAs) were introduced, providing the possibility for rapid single sample testing. More and more LFAs are entering the market, but, although often being equated, all use their own antibodies, procedures and interpretation criteria. A recent European survey revealed that about 24-33% of laboratories implemented a lateral flow assay on-site. METHODS: We conducted a survey at 81 Belgian hospital laboratories regarding the implementation of LFAs in their centre. In addition, we performed an extensive review of all publicly available studies on the performance of lateral flow assays to diagnose invasive aspergillosis. RESULTS: Response rate to the survey was 69%. Of the 56 responding hospital laboratories, 6 (11%) used an LFA. The Soña Aspergillus galactomannan LFA (IMMY, Norman, Oklahoma, USA) was used in 4/6 centres, while two centres used the QuicGM (Dynamiker, Tianjin, China) and one centre used the FungiXpert Aspergillus Galactomannan Detection K-set LFA (Genobio [Era Biology Technology], Tianjin, China). One centre used 2 distinct LFAs. In 3/6 centres, the sample is sent to another lab for confirmation with GM-EIA when the LFA result is positive and in 2/6 when the LFA results is negative. In one centre, a confirmatory GM-EIA is always performed in house. In three centres the LFA result is used as a complete substitute for GM-EIA. Available LFA performance studies are very diverse and results vary in function of the study population and type of LFA. Apart from the IMMY and OLM LFA, only very limited performance data are available. From two out of three LFAs used in Belgium, no clinical performance studies are published in literature. CONCLUSIONS: A large variety of LFAs are used in Belgian Hospitals, some of which no clinical validation studies are published. These results do likely have implications for other parts of Europe and for the rest of the world as well. Due to the variable performance of LFA tests and the limited validation data available, each laboratory must check the available performance information of the specific test considered for implementation. In addition, laboratories should perform an implementation verification study.

Performance of the JF5-Based Galactomannoprotein EIA Compared to the Lateral Flow Device and the Galactomannan EIA in Serum and Bronchoalveolar Lavage Fluid.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of "probable" invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4-deviating from the 0.5 threshold recommended by the manufacturer-sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

A novel rRNA hybridization-based approach to rapid, accurate Candida identification directly from blood culture.

Invasive fungal infections are increasingly common and carry high morbidity and mortality, yet fungal diagnostics lag behind bacterial diagnostics in rapidly identifying the causal pathogen. We previously devised a fluorescent hybridization-based assay to identify bacteria within hours directly from blood culture bottles without subculture, called phylogeny-informed rRNA-based strain identification (Phirst-ID). Here, we adapt this approach to unambiguously identify 11 common pathogenic Candida species, including C. auris, with 100% accuracy from laboratory culture (33 of 33 strains in a reference panel, plus 33 of 33 additional isolates tested in a validation panel). In a pilot study on 62 consecutive positive clinical blood cultures from two hospitals that showed yeast on Gram stain, Candida Phirst-ID matched the clinical laboratory result for 58 of 59 specimens represented in the 11-species reference panel, without misclassifying the 3 off-panel species. It also detected mixed Candida species in 2 of these 62 specimens, including the one discordant classification, that were not identified by standard clinical microbiology workflows; in each case the presence of both species was validated by both clinical and experimental data. Finally, in three specimens that grew both bacteria and yeast, we paired our prior bacterial probeset with this new Candida probeset to detect both pathogen types using Phirst-ID. This simple, robust assay can provide accurate Candida identification within hours directly from blood culture bottles, and the conceptual approach holds promise for pan-microbial identification in a single workflow. LAY SUMMARY: Candida bloodstream infections cause considerable morbidity and mortality, yet slow diagnostics delay recognition, worsening patient outcomes. We develop and validate a novel molecular approach to accurately identify Candida species directly from blood culture one day faster than standard workflows.

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.

Elevated Aspergillus-specific antibody levels among HIV infected Ugandans with pulmonary tuberculosis.

BACKGROUND: The incidence of tuberculosis (TB) is high among human immunodeficiency virus (HIV) infected Ugandans. Recent evidence suggests that Chronic Pulmonary Aspergillosis and Aspergillus sensitisation might be responsible for significant mortality in patients treated for tuberculosis in Uganda. METHODS: We retrieved and tested paired serum aliquots for 101 HIV-TB co-infected patients at the beginning and week 24 of TB treatment. We tested samples for Aspergillus-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) using ImmunoCAP®; and Aspergillus-specific IgG and total serum IgE using Immulite® immunoassays. We compared antibody levels between baseline and week 24, relating them to selected baseline characteristics. RESULTS: 10% of the patients had elevated Aspergillus-specific IgE (Aspergillus sensitization) and Aspergillus-specific IgG antibodies were elevated in 9% of the patients at the end of TB treatment. There was a significant fall in the Aspergillus-specific IgG antibody levels between baseline and week 24 (P = 0.02). Patients with cluster of differentiation 4 (CD4) T-cell count <100 cells/µl and those who were not on anti-retroviral therapy at baseline had more elevated Aspergillus-specific IgG antibodies (P = 0.01, P = 0.03). The ImmunoCAP® Aspergillus-specific IgG antibody titres were higher at week 24 than baseline with more positives at week 24; even though the difference in means was small. However, this difference was statistically significant (P = 0.02). Pulmonary infiltrates were the commonest x-ray abnormality and only 5% of the patients had pulmonary cavities on chest x-ray at week 24. CONCLUSION: These results suggest that Aspergillus infection may complicate active pulmonary TB and further studies including fungal culture and thoracic imaging may now be indicated to measure the prevalence of pulmonary aspergillosis complicating tuberculosis. TRIAL REGISTRATION: The SOUTH trial was registered prospectively. ClinicalTrials.gov Identifier: NCT01782950 ; Registration date: 4th February 2013; Last verified: 13th April 2015.

Executive Summary: Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

Executive Summary: Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

Distinct galactofuranose antigens in the cell wall and culture supernatants as a means to differentiate Fusarium from Aspergillus species.

Detection of carbohydrate antigens is an important means for diagnosis of invasive fungal infections. For diagnosis of systemic Aspergillus infections, galactomannan is commonly used, the core antigenic structure of which consists of chains of several galactofuranose moieties. In this study, we provide evidence that Fusarium produces at least two distinct galactofuranose antigens: Smaller amounts of galactomannan and larger quantities of a novel antigen recognized by the monoclonal antibody AB135-8. In A. fumigatus, only minor amounts of the AB135-8 antigen are found in supernatants and in the apical regions of hyphae. A galactofuranose-deficient A. fumigatus mutant lacks the AB135-8 antigen, which strongly suggests that galactofuranose is an essential constituent of this antigen. Using a combination of AB135-8 and a galactomannan-specific antibody, we were able to unambiguously differentiate A. fumigatus and Fusarium hyphae in immunohistology. Moreover, since Fusarium releases the AB135-8 antigen, it appears to be a promising target antigen for a serological detection of Fusarium infections.

Comparison of different lateral flow assays on bronchoalveolar lavage fluid for invasive aspergillosis screening in non-hematological patients.

Several lateral flow assays (LFA) capable of detecting Aspergillus fumigatus in serum and broncho-alveolar lavage fluid (BALF) within the hour, thereby potentially accelerating the screening process, are now commercially available. We prospectively compared three LFA targeting A. fumigatus on BALF collected from non-surgical intensive care patients between June 2022 and February 2023. The three LFA tested were Sõna Aspergillus galactomannan LFA (Immy), Fungadia Aspergillus antigen (Gadia), and AspLFD (OLM Diagnostics). We compared the results of these LFA with those of the galactomannan (GM) Platelia Aspergillus enzyme immunoassay (Bio-Rad), culture on Sabouraud medium and Aspergillus qPCR. We tested 97 BALF samples from 92 patients. In total 84 BALF samples tested negative with all three LFA, and four BALF samples tested positive with the AspLFD assay only (OLM). Only one BALF sample tested positive with the three LFA. In addition, three BALF samples tested positive only with the GM Platelia immunoassay. Four diagnosis of probable invasive aspergillosis were retained for the 92 patients tested. This prospective series included very few positive samples. From a practical point of view, the LFA from OLM presented the simplest protocol for use.

The Impact of the Fungal Priority Pathogens List on Medical Mycology: A Northern European Perspective.

Fungal diseases represent a considerable global health concern, affecting >1 billion people annually. In response to this growing challenge, the World Health Organization introduced the pivotal fungal priority pathogens list (FPPL) in late 2022. The FPPL highlights the challenges in estimating the global burden of fungal diseases and antifungal resistance (AFR), as well as limited surveillance capabilities and lack of routine AFR testing. Furthermore, training programs should incorporate sufficient information on fungal diseases, necessitating global advocacy to educate health care professionals and scientists. Established international guidelines and the FPPL are vital in strengthening local guidance on tackling fungal diseases. Future iterations of the FPPL have the potential to refine the list further, addressing its limitations and advancing our collective ability to combat fungal diseases effectively. Napp Pharmaceuticals Limited (Mundipharma UK) organized a workshop with key experts from Northern Europe to discuss the impact of the FPPL on regional clinical practice.

Shared Vision for Improving Outcomes for Serious Fungal Diseases: Report of a Patient, Caregiver, and Clinician Summit.

Background: Recently, increasing focus on patient input into research and healthcare improvements has fostered expanded patient-centered advocacy efforts. This first pan-fungal disease summit, part of the MYCology Advocacy, Research, & Education effort, brought together patients, caregivers, and mycology experts to better document patient experiences with invasive fungal disease (IFD) and establish priorities for mycology education, advocacy, and research. Methods: Patients who had suffered from IFD, their caregivers, clinicians, industry representatives, government officials, and patient advocacy professionals were invited. Patients and caregivers shared their stories and struggles with IFD. Breakout sessions separated mycology experts from patients and caregivers for further discussions to identify commonalities and perceived gaps and to formulate recommendations. The 2 groups then reconvened to develop consensus recommendations. Results: IFD patients and their caregivers shared experiences reflecting the typically lengthy prediagnosis, acute treatment, long-term treatment, and posttreatment recovery stages of IFD. They reported substantial physical, psychological, and financial burdens associated with the IFD experience, particularly related to delayed diagnoses. They reaffirmed a need for coordinated patient-centered education, peer support, and advocacy to document the burden of serious fungal infections. Mycology experts discussed strategies to address gaps in the mycology field, such as insufficient training, inadequate workforce support, and a need to partner more with patient groups. Conclusions: A summit involving patients with IFD, family caregivers, and mycology experts identified a substantial nonclinical burden of disease associated with IFD. Patients and mycology experts prioritized several goals for education, advocacy, and research to raise awareness of IFD and improve outcomes.

The diagnostic and clinical utility of microbial cell-free DNA sequencing in a real-world setting.

Microbial cell free DNA sequencing is increasingly used for diagnosis of infection but few studies describe its utility in real-world settings. We performed a single-center retrospective case series of microbial cell free DNA testing using the Karius assay from 29 patient samples to define the clinical reasoning and the impact of testing. Indications fell into 3 categories, identifying a causative pathogen in patients with an infectious syndrome and negative microbiologic workup (15/29, 52%), seeking another pathogen when organisms identified by traditional diagnostics failed to explain the clinical presentation (9/29, 31%) and to "rule out" infection in patients with nonspecific symptoms and negative microbiologic workup (5/29, 17%). Clinical impact was positive in 13/29 (45%) and all were for patients with high pretest probability for infection. Impact was negative in 3/29 (10%) cases. There was no impact in 15/29 (52%) cases. Further work is needed to define the optimal timing accounting for test performance, and patient characteristics.

The Novel Monoclonal IgG1-Antibody AB90-E8 as a Diagnostic Tool to Rapidly Distinguish Aspergillus fumigatus from Other Human Pathogenic Aspergillus Species.

In most cases, invasive aspergillosis (IA) is caused by A. fumigatus, though infections with other Aspergillus spp. with lower susceptibilities to amphotericin B (AmB) gain ground. A. terreus, for instance, is the second leading cause of IA in humans and of serious concern because of its high propensity to disseminate and its in vitro and in vivo resistance to AmB. An early differentiation between A. fumigatus and non-A. fumigatus infections could swiftly recognize a potentially ineffective treatment with AmB and lead to the lifesaving change to a more appropriate drug regime in high-risk patients. In this study, we present the characteristics of the monoclonal IgG1-antibody AB90-E8 that specifically recognizes a surface antigen of A. fumigatus and the closely related, but not human pathogenic A. fischeri. We show immunostainings on fresh frozen sections as well as on incipient mycelium picked from agar plates with tweezers or by using the expeditious tape mount technique. All three methods have a time advantage over the common procedures currently used in the routine diagnosis of IA and outline the potential of AB90-E8 as a rapid diagnostic tool.

Point-of-Care Testing for the Diagnosis of Fungal Infections: Current Testing Applications and Potential for the Future.

Invasive fungal infections are increasing worldwide due to factors such as climate change and immunomodulating therapies. Unfortunately, the detection of these infections is limited due to the low sensitivity and long periods required for laboratory testing. Point-of-care testing could lead to more rapid diagnosis of these often devasting infections. However, there are currently no true point-of-care tests on the market for the detection of fungi. In this article, the current state of fungal antigen and molecular testing is reviewed, with commentary on the potential for development and use in the point-of-care setting.

Teaching Everyone Everywhere All at Once: Leveraging Social Media to Implement a Multisite Fungal Diagnostics Curriculum.

Background: Environmental fungi are threats to personal and public health. Fungal in vitro diagnostics help diagnose invasive fungal infections (IFIs), but clinicians remain underinformed about their use and interpretation. Given the increasing use of social media to share infectious diseases-related content, we designed and implemented a multisite Twitter-based curriculum focused on IFIs and related diagnostics. Methods: Questions were posted through a dedicated Twitter account twice weekly over 8 weeks. We surveyed clinicians at 3 US academic centers before and after completion of the curriculum and interviewed a subset of participants. We undertook quantitative and qualitative evaluations and reviewed Twitter analytics. Results: We surveyed 450 participants. One hundred twenty-one participants (27%) completed the knowledge assessment precurriculum, 68 (15%) postcurriculum, and 53 (12%) pre- and postcurriculum. We found a significant increase (72% vs 80%, P = .005) in the percentage of correct answers in the pre- versus postcurriculum knowledge assessments. Perceived benefits included a well-executed curriculum that facilitated engagement with appropriately detailed tweetorials from a dedicated Twitter account. Perceived barriers included lack of awareness of tweetorial posts and timing, competing priorities, and the coronavirus disease 2019 pandemic. The Twitter account accrued 1400 followers from 65 countries during the 8-week period. Tweets with multiple-choice questions had a median of 14 904 impressions (interquartile range [IQR], 12 818-16 963), 798 engagements (IQR, 626-1041), and an engagement rate of 6.1% (IQR, 4.2%-6.6%). Conclusions: Educators can leverage social media to share content with a large audience and improve knowledge while being mindful of the barriers associated with implementing a curriculum on social media.

Comparison of Indirect Fungal Diagnostic Tests in Patients With Proven Histoplasmosis.

Background: Histoplasmosis is a common cause of invasive fungal infection in endemic regions and accurate diagnosis is difficult without direct tissue culture or pathology. Indirect fungal antigen testing for various fungal pathogens are typically performed to assist with diagnostic workup, though cross-reaction can lead to difficulty in interpreting results. We aimed to compare indirect fungal diagnostic tests and evaluate prevalence of positive antigen testing for non-Histoplasma fungal pathogens in patients with proven histoplasmosis. Methods: We performed a single-center retrospective review of adult patients with proven histoplasmosis diagnosed by fungal culture and/or cytology from January 2010 to March 2018. Patient demographics, clinical characteristics, and results of fungal antigen testing for Histoplasma, Blastomyces, Aspergillus, Cryptococcus, and (1→3)-ß-D-glucan were evaluated. Two different urine Histoplasma antigen assays were used during the study period. Results: Fifty-seven of 182 (31.3%) patients reviewed had proven histoplasmosis and presented with acute pulmonary (n = 10), chronic pulmonary (n = 7), and disseminated (n = 40) disease. Forty-one (72%) of these patients were immunosuppressed. Urine Blastomyces antigen (93%) and serum (1→3)-ß-D-glucan (88%) were commonly positive in patients with histoplasmosis, whereas Aspergillus antigen was detected in 50% of patients and Cryptococcus antigenemia was rare (5%). In patients with disseminated disease, the MiraVista urine Histoplasma antigen assay had higher sensitivity than the Viracor urine Histoplasma antigen assay (86% vs 50%, respectively; P = .019). Conclusions: Noninvasive fungal antigen assays are helpful diagnostic tools; however, given their low specificity, clinicians must be aware of the various clinical presentations of invasive fungal infections and be aware of the limitations of these tests.