Universal digital high-resolution melting for the detection of pulmonary mold infections.

Invasive mold infections (IMIs) are associated with high morbidity, particularly in immunocompromised patients, with mortality rates between 40% and 80%. Early initiation of appropriate antifungal therapy can substantially improve outcomes, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive mycological findings. Universal digital high-resolution melting (U-dHRM) analysis may enable rapid and robust diagnoses of IMI. A universal fungal assay was developed for U-dHRM and used to generate a database of melt curve signatures for 19 clinically relevant fungal pathogens. A machine learning algorithm (ML) was trained to automatically classify these pathogen curves and detect novel melt curves. Performance was assessed on 73 clinical bronchoalveolar lavage samples from patients suspected of IMI. Novel curves were identified by micropipetting U-dHRM reactions and Sanger sequencing amplicons. U-dHRM achieved 97% overall fungal organism identification accuracy and a turnaround time of ~4 hrs. U-dHRM detected pathogenic molds (Aspergillus, Mucorales, Lomentospora, and Fusarium) in 73% of 30 samples classified as IMI, including mixed infections. Specificity was optimized by requiring the number of pathogenic mold curves detected in a sample to be >8 and a sample volume to be 1 mL, which resulted in 100% specificity in 21 at-risk patients without IMI. U-dHRM showed promise as a separate or combination diagnostic approach to standard mycological tests. U-dHRM's speed, ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples, and detect emerging opportunistic pathogens may aid treatment decisions, improving patient outcomes. IMPORTANCE: Improvements in diagnostics for invasive mold infections are urgently needed. This work presents a new molecular detection approach that addresses technical and workflow challenges to provide fast pathogen detection, identification, and quantification that could inform treatment to improve patient outcomes.

Age difference of patients with and without invasive aspergillosis: a systematic review and meta-analysis.

BACKGROUND: Invasive Aspergillosis (IA) is a life-threatening fungal disease with significant mortality rates. Timely diagnosis and treatment greatly enhance patient outcomes. This study aimed to explore the association between patient age and the development of IA, as well as the potential implications for risk stratification strategies. METHODS: We searched National Center for Biotechnology Information (NCBI) databases for publications until October 2023 containing age characteristics of patients with and without IA. A random-effects model with the application of inverse-variance weighting was used to pool reported estimates from each study, and meta-regression and subgroup analyses were utilized to assess sources of heterogeneity. RESULTS: A systematic review was conducted, resulting in the inclusion of 55 retrospective observational studies with a total of 13,983 patients. Meta-analysis revealed that, on average, patients with IA were approximately two and a half years older (95% Confidence Interval [CI] 1.84-3.31 years; I2 = 26.1%) than those without the disease (p < 0.0001). No significant moderators could explain the observed heterogeneity in age difference. However, subgroup analysis revealed that age differences were more pronounced within particular patient groups compared to others. For example, patients with and without IA who had primary severe lung infections exhibited a greater difference in mean age than other patient cohorts. CONCLUSIONS: Further research, such as individual patient data meta-analysis, is necessary to better understand the potential relationship between increasing age and the likelihood of IA. Improved risk stratification strategies based on patient age could potentially enhance the early detection and treatment of IA, ultimately improving patient outcomes.

Universal Digital High Resolution Melt for the detection of pulmonary mold infections.

Background: Invasive mold infections (IMIs) such as aspergillosis, mucormycosis, fusariosis, and lomentosporiosis are associated with high morbidity and mortality, particularly in immunocompromised patients, with mortality rates as high as 40% to 80%. Outcomes could be substantially improved with early initiation of appropriate antifungal therapy, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive mycological findings. Universal digital high resolution melting analysis (U-dHRM) may enable rapid and robust diagnosis of IMI. This technology aims to accomplish timely pathogen detection at the single genome level by conducting broad-based amplification of microbial barcoding genes in a digital polymerase chain reaction (dPCR) format, followed by high-resolution melting of the DNA amplicons in each digital reaction to generate organism-specific melt curve signatures that are identified by machine learning. Methods: A universal fungal assay was developed for U-dHRM and used to generate a database of melt curve signatures for 19 clinically relevant fungal pathogens. A machine learning algorithm (ML) was trained to automatically classify these 19 fungal melt curves and detect novel melt curves. Performance was assessed on 73 clinical bronchoalveolar lavage (BAL) samples from patients suspected of IMI. Novel curves were identified by micropipetting U-dHRM reactions and Sanger sequencing amplicons. Results: U-dHRM achieved an average of 97% fungal organism identification accuracy and a turn-around-time of 4hrs. Pathogenic molds (Aspergillus, Mucorales, Lomentospora and Fusarium) were detected by U-dHRM in 73% of BALF samples suspected of IMI. Mixtures of pathogenic molds were detected in 19%. U-dHRM demonstrated good sensitivity for IMI, as defined by current diagnostic criteria, when clinical findings were also considered. Conclusions: U-dHRM showed promising performance as a separate or combination diagnostic approach to standard mycological tests. The speed of U-dHRM and its ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples as well as detect emerging opportunistic pathogens may provide information that could aid in treatment decisions and improve patient outcomes.

An Evaluation of the OLM PneumID Real-Time Polymerase Chain Reaction to Aid in the Diagnosis of Pneumocystis Pneumonia.

BACKGROUND: The use of the PCR to aid in the diagnosis of Pneumocystis pneumonia (PcP) has demonstrated excellent clinical performance, as evidenced through various systematic reviews and meta-analyses, yet there are concerns over the interpretation of positive results due to the potential presence of Pneumocystis colonization of the airways. While this can be overcome by applying designated positivity thresholds to PCR testing, the shear number of assays described limits the development of a universal threshold. Commercial assays provide the opportunity to overcome this problem, provided satisfactory performance is determined through large-scale, multi-centre evaluations. METHODS: Retrospective case/control and consecutive cohort performance evaluations of the OLM PneumID real-time PCR assay were performed on DNA eluates from a range of samples sent from patients where "in-house" PCR had been performed as part of routine diagnostic testing. The clinical performance of the PneumID assay was determined before including it in a diagnostic algorithm to provide the probability of PcP (dependent on diagnostic evidence). RESULTS: After being used to test 317 patients (32 with PcP), the overall performance of the PneumID assay was found to be excellent (Sensitivity/Specificity: 96.9%/95.1%). False positivity could be removed by applying a threshold specific to sample type (<33.1 cycles for BAL fluid; <37.0 cycles for throat swabs), whereas considering any positive respiratory samples as significant generated 100% sensitivity, making absolute negativity sufficient to exclude PcP. Incorporating the PneumID assay into diagnostic algorithms alongside (1-3)-ß-D-Glucan testing provided high probabilities of PcP (up to 85.2%) when both were positive and very low probabilities (<1%) when both were negative. CONCLUSIONS: The OLM PneumID qPCR provides a commercial option for the accurate diagnosis of PcP, generating excellent sensitivity and specificity, particularly when testing respiratory specimens. The combination of PcP PCR with serum (1-3)-ß-D-Glucan provides excellent clinical utility for diagnosing PcP.

An Overview of Systematic Reviews of Polymerase Chain Reaction (PCR) for the Diagnosis of Invasive Aspergillosis in Immunocompromised People: A Report of the Fungal PCR Initiative (FPCRI)-An ISHAM Working Group.

This overview of reviews (i.e., an umbrella review) is designed to reappraise the validity of systematic reviews (SRs) and meta-analyses related to the performance of Aspergillus PCR tests for the diagnosis of invasive aspergillosis in immunocompromised patients. The methodological quality of the SRs was assessed using the AMSTAR-2 checklist; the quality of the evidence (QOE) within each SR was appraised following the GRADE approach. Eight out of 12 SRs were evaluated for qualitative and quantitative assessment. Five SRs evaluated Aspergillus PCR on bronchoalveolar lavage fluid (BAL) and three on blood specimens. The eight SRs included 167 overlapping reports (59 evaluating PCR in blood specimens, and 108 in BAL), based on 107 individual primary studies (98 trials with a cohort design, and 19 with a case-control design). In BAL specimens, the mean sensitivity and specificity ranged from 0.57 to 0.91, and from 0.92 to 0.97, respectively (QOE: very low to low). In blood specimens (whole blood or serum), the mean sensitivity ranged from 0.57 to 0.84, and the mean specificity from 0.58 to 0.95 (QOE: low to moderate). Across studies, only a low proportion of AMSTAR-2 critical domains were unmet (1.8%), demonstrating a high quality of methodological assessment. Conclusions. Based on the overall methodological assessment of the reviews included, on average we can have high confidence in the quality of results generated by the SRs.

An update on current and novel molecular diagnostics for the diagnosis of invasive fungal infections.

BACKGROUND: Invasive fungal infections cause millions of infections annually, but diagnosis remains challenging. There is an increased need for low-cost, easy to use, highly sensitive and specific molecular assays that can differentiate between colonized and pathogenic organisms from different clinical specimens. AREAS COVERED: We reviewed the literature evaluating the current state of molecular diagnostics for invasive fungal infections, focusing on current and novel molecular tests such as polymerase chain reaction (PCR), digital PCR, high-resolution melt (HRM), and metagenomics/next generation sequencing (mNGS). EXPERT OPINION: PCR is highly sensitive and specific, although performance can be impacted by prior/concurrent antifungal use. PCR assays can identify mutations associated with antifungal resistance, non-Aspergillus mold infections, and infections from endemic fungi. HRM is a rapid and highly sensitive diagnostic modality that can identify a wide range of fungal pathogens, including down to the species level, but multiplex assays are limited and HRM is currently unavailable in most healthcare settings, although universal HRM is working to overcome this limitation. mNGS offers a promising approach for rapid and hypothesis-free diagnosis of a wide range of fungal pathogens, although some drawbacks include limited access, variable performance across platforms, the expertise and costs associated with this method, and long turnaround times in real-world settings.

COVID-19-associated pulmonary aspergillosis in mechanically ventilated patients: a prospective, multicentre UK study.

BACKGROUND: Invasive pulmonary aspergillosis is a complication of severe COVID-19, with regional variation in reported incidence and mortality. We describe the incidence, risk factors and mortality associated with COVID-19-associated pulmonary aspergillosis (CAPA) in a prospective, multicentre UK cohort. METHODS: From March 2020 to March 2021, 266 mechanically ventilated adults with COVID-19 were enrolled across 5 UK hospital intensive care units (ICUs). CAPA was defined using European Confederation for Medical Mycology and the International Society for Human and Animal Mycology criteria and fungal diagnostics performed on respiratory and serum samples. RESULTS: Twenty-nine of 266 patients (10.9%) had probable CAPA, 14 (5.2%) possible CAPA and none proven CAPA. Probable CAPA was diagnosed a median of 9 (IQR 7-16) days after ICU admission. Factors associated with probable CAPA after multivariable logistic regression were cumulative steroid dose given within 28 days prior to ICU admission (adjusted OR (aOR) 1.16; 95% CI 1.01 to 1.43 per 100 mg prednisolone-equivalent), receipt of an interleukin (IL)-6 inhibitor (aOR 2.79; 95% CI 1.22 to 6.48) and chronic obstructive pulmonary disease (COPD) (aOR 4.78; 95% CI 1.13 to 18.13). Mortality in patients with probable CAPA was 55%, vs 46% in those without. After adjustment for immortal time bias, CAPA was associated with an increased risk of 90-day mortality (HR 1.85; 95% CI 1.07 to 3.19); however, this association did not remain statistically significant after further adjustment for confounders (adjusted HR 1.57; 95% CI 0.88 to 2.80). There was no difference in mortality between patients with CAPA prescribed antifungals (9 of 17; 53%) and those who were not (7 of 12; 58%) (p=0.77). INTERPRETATION: In this first prospective UK study, probable CAPA was associated with corticosteroid use, receipt of IL-6 inhibitors and pre-existing COPD. CAPA did not impact mortality following adjustment for prognostic variables.

Predictors for Prolonged Hospital Stay Solely to Complete Intravenous Antifungal Treatment in Patients with Candidemia: Results from the ECMM Candida III Multinational European Observational Cohort Study.

BACKGROUND: To date, azoles represent the only viable option for oral treatment of invasive Candida infections, while rates of azole resistance among non-albicans Candida spp. continue to increase. The objective of this sub-analysis of the European multicenter observational cohort study Candida III was to describe demographical and clinical characteristics of the cohort requiring prolonged hospitalization solely to complete intravenous (iv) antifungal treatment (AF Tx). METHODS: Each participating hospital (number of eligible hospitals per country determined by population size) included the first ~ 10 blood culture proven adult candidemia cases occurring consecutively after July 1st, 2018, and treating physicians answered the question on whether hospital stay was prolonged only for completion of intravenous antifungal therapy. Descriptive analyses as well as binary logistic regression was used to assess for predictors of prolonged hospitalization solely to complete iv AF Tx. FINDINGS: Hospital stay was prolonged solely for the completion of iv AF Tx in 16% (100/621) of candidemia cases by a median of 16 days (IQR 8 - 28). In the multivariable model, initial echinocandin treatment was a positive predictor for prolonged hospitalization to complete iv AF Tx (aOR 2.87, 95% CI 1.55 - 5.32, p < 0.001), while (i) neutropenia, (ii) intensive care unit admission, (iii) catheter related candidemia, (iv) total parenteral nutrition, and (v) C. parapsilosis as causative pathogen were found to be negative predictors (aOR 0.22 - 0.45; p < 0.03). INTERPRETATION: Hospital stays were prolonged due to need of iv AF Tx in 16% of patients with candidemia. Those patients were more likely to receive echinocandins as initial treatment and were less severely ill and less likely infected with C. parapsilosis.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study.

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

Developments in Fungal Serology.

Purpose of Review: The true incidence of fungal disease is hampered by conventionally poor diagnostic tests, limited access to advanced diagnostics, and limited surveillance. The availability of serological testing has been available for over two decades and generally underpins the modern diagnosis of the most common forms of fungal disease. This review will focus on technical developments of serological tests for the diagnosis of fungal disease, describing advances in clinical performance when available. Recent Findings: Despite their longevity, technical, clinical, and performance limitations remain, and tests specific for fungal pathogens outside the main pathogens are lacking. The availability of LFA and automated systems, capable of running multiple different tests, represents significant developments, but clinical performance data is variable and limited. Summary: Fungal serology has significantly advanced the diagnosis of the main fungal infections, with LFA availability increasing accessibility to testing. Combination testing has the potential to overcome performance limitations.

Gaining an Understanding of Pneumocystosis in Wales.

Pneumocystis pneumonia (PcP) is a serious complication of many significant immunocompromising conditions. Prior incidence estimates in Wales are based on PcP's presentation in the HIV and transplant populations. The objectives were to describe the incidence of PcP in Wales using laboratory reporting measures and assess the impact of underlying immunosuppression cause on mortality. All positive PCR results for PcP between 2015 and 2018 were identified. The total number of unique positives with clinical and radiological correlation was 159 patients, a mean of 39.75 annually. The healthcare records of these patients were reviewed. The mortality at one month was 35.2% and 49.1% at one year. HIV remains the commonest cause of immunosuppression but has lower mortality than non-HIV conditions (12% vs. 59% at one year, p < 0.00001). Non-HIV conditions were categorised as life-threatening and non-life threatening but had a non-significant mortality (66% vs. 54%; p = 0.149), highlighting the negative impact of PcP. An incidence of PcP in Wales of 1.23-1.26 cases per 100,000 has been identified, 32-35% greater than the upper limit previously estimated. There is high mortality in non-HIV patients regardless of immunosuppression cause. A heightened awareness of PcP in these groups will hasten diagnosis and potentially improve mortality.

Metagenomic Next-Generation Sequencing of Plasma for Diagnosis of COVID-19-Associated Pulmonary Aspergillosis.

Timely diagnosis remains an unmet need in non-neutropenic patients at risk for aspergillosis, including those with COVID-19-associated pulmonary aspergillosis (CAPA), which in its early stages is characterized by tissue-invasive growth of the lungs with limited angioinvasion. Currently available mycological tests show limited sensitivity when testing blood specimens. Metagenomic next-generation sequencing (mNGS) to detect microbial cell-free DNA (mcfDNA) in plasma might overcome some of the limitations of conventional diagnostics. A two-center cohort study involving 114 COVID-19 intensive care unit patients evaluated the performance of plasma mcfDNA sequencing for the diagnosis of CAPA. Classification of CAPA was performed using the European Confederation for Medical Mycology (ECMM)/International Society for Human and Animal Mycoses (ISHAM) criteria. A total of 218 plasma samples were collected between April 2020 and June 2021 and tested for mcfDNA (Karius test). Only 6 patients were classified as probable CAPA, and 2 were classified as possible, while 106 patients did not fulfill CAPA criteria. The Karius test detected DNA of mold pathogens in 12 samples from 8 patients, including Aspergillus fumigatus in 10 samples from 6 patients. Mold pathogen DNA was detected in 5 of 6 (83% sensitivity) cases with probable CAPA (A. fumigatus in 8 samples from 4 patients and Rhizopus microsporus in 1 sample), while the test did not detect molds in 103 of 106 (97% specificity) cases without CAPA. The Karius test showed promising performance for diagnosis of CAPA when testing plasma, being highly specific. The test detected molds in all but one patient with probable CAPA, including cases where other mycological tests from blood resulted continuously negative, outlining the need for validation in larger studies.

The Use of Host Biomarkers for the Management of Invasive Fungal Disease.

Invasive fungal disease (IFD) causes severe morbidity and mortality, and the number of IFD cases is increasing. Exposure to opportunistic fungal pathogens is inevitable, but not all patients with underlying diseases increasing susceptibility to IFD, develop it. IFD diagnosis currently uses fungal biomarkers and clinical risk/presentation to stratify high-risk patients and classifies them into possible, probable, and proven IFD. However, the fungal species responsible for IFD are highly diverse and present numerous diagnostic challenges, which culminates in the empirical anti-fungal treatment of patients at risk of IFD. Recent studies have focussed on host-derived biomarkers that may mediate IFD risk and can be used to predict, and even identify IFD. The identification of novel host genetic variants, host gene expression changes, and host protein expression (cytokines and chemokines) associated with increased risk of IFD has enhanced our understanding of why only some patients at risk of IFD actually develop disease. Furthermore, these host biomarkers when incorporated into predictive models alongside conventional diagnostic techniques enhance predictive and diagnostic results. Once validated in larger studies, host biomarkers associated with IFD may optimize the clinical management of populations at risk of IFD. This review will summarise the latest developments in the identification of host biomarkers for IFD, their use in predictive modelling and their potential application/usefulness for informing clinical decisions.

Defective antifungal immunity in patients with COVID-19.

The COVID-19 pandemic has placed a huge strain on global healthcare and been a significant cause of increased morbidity and mortality, particularly in at-risk populations. This disease attacks the respiratory systems and causes significant immune dysregulation in affected patients creating a perfect opportunity for the development of invasive fungal disease (IFD). COVID-19 infection can instill a significant, poorly regulated pro-inflammatory response. Clinically induced immunosuppression or pro-inflammatory damage to mucosa facilitate the development of IFD and Aspergillus, Mucorales, and Candida infections have been regularly reported throughout the COVID-19 pandemic. Corticosteroids and immune modulators are used in the treatment of COVID-19. Corticosteroid use is also a risk factor for IFD, but not the only reason for IFD in COVID -19 patients. Specific dysregulation of the immune system through functional exhaustion of Natural killer (NK) cells and T cells has been observed in COVID-19 through the expression of the exhaustion markers NK-G2A and PD-1. Reduced fungicidal activity of neutrophils from COVID-19 patients indicates that immune dysfunction/imbalance are important risk factors for IFD. The COVID-19 pandemic has significantly increased the at-risk population for IFD. Even if the incidence of IFD is relatively low, the size of this new at-risk population will result in a substantial increase in the overall, annual number of IFD cases. It is important to understand how and why certain patients with COVID-19 developed increased susceptibility to IFD, as this will improve our understanding of risk of IFD in the face of future pandemics but also in a clinical era of increased clinical immuno-suppression/modulation.

Dual use of antifungals in medicine and agriculture: How do we help prevent resistance developing in human pathogens?

Azole resistance in Aspergillus fumigatus is a One Health resistance threat, where azole fungicide exposure compromises the efficacy of medical azoles. The use of the recently authorized fungicide ipflufenoquin, which shares its mode-of-action with a new antifungal olorofim, underscores the need for risk assessment for dual use of antifungals.

COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds.

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus- and R. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients.

CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models.

Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.

An Evaluation of the OLM CandID Real-Time PCR to Aid in the Diagnosis of Invasive Candidiasis When Testing Serum Samples.

Background: Treatment for invasive candidiasis (IC) is time-critical, and culture-based tests can limit clinical utility. Nonculture-based methods such as Candida PCR represent a promising approach to improving patient management but require further evaluation to understand their optimal role and incorporation into clinical algorithms. This study determined the performance of the commercially available OLM CandID real-time PCR when testing serum and developed a diagnostic algorithm for IC. Methods: The study comprised a retrospective performance evaluation of the CandID real-time PCR assay when testing surplus serum (n = 83 patients, 38 with IC), followed by a prospective consecutive cohort evaluation (n = 103 patients, 24 with IC) post incorporation into routine service. A combined diagnostic algorithm, also including (1-3)-ß-D-Glucan testing, was generated. Results: Prospective CandID testing generated a sensitivity/specificity of 88%/82%, respectively. Specificity was improved (>95%) when both PCR replicates were positive and/or the patient had multiple positive samples. When combining CandID with (1-3)-ß-D-Glucan testing, the probability of IC when both were positive or negative was >69% or <1%, respectively. Conclusions: The CandID provides excellent performance and a rapid time-to-result using methods widely available in generic molecular diagnostic laboratories. By combining nonculture diagnostics, it may be possible to accurately confirm or exclude IC.

CT findings of COVID-19-associated pulmonary aspergillosis: a systematic review and individual patient data analysis.

PURPOSE: Common CT abnormalities of pulmonary aspergillosis represent a cavity with air-meniscus sign, nodule, mass, and consolidation having an angio-invasive pattern. This study aims to conduct a systematic review and an individual patient-level image analysis of CT findings of COVID-19-associated pulmonary aspergillosis (CAPA). METHODS: A systematic literature search was conducted to identify studies reporting CT findings of CAPA as of January 7, 2021. We summarized study-level clinical and CT findings of CAPA and collected individual patient CT images by inviting corresponding authors. The CT findings were categorized into four groups: group 1, typical appearance of COVID-19; group 2, indeterminate appearance of COVID-19; group 3, atypical for COVID-19 without cavities; and group 4, atypical for COVID-19 with cavities. In group 2, cases had only minor discrepant findings including solid nodules, isolated airspace consolidation with negligible ground-glass opacities, centrilobular micronodules, bronchial abnormalities, and cavities. RESULTS: The literature search identified 89 patients from 25 studies, and we collected CT images from 35 CAPA patients (mean age 62.4 ± 14.6 years; 21 men): group 1, thirteen patients (37.1%); group 2, eight patients (22.9%); group 3, six patients (17.1%); and group 4, eight patients (22.9%). Eight of the 14 patients (57.1%) with an atypical appearance had bronchial abnormalities, whereas only one (7.1%) had an angio-invasive fungal pattern. In the study-level analysis, cavities were reported in 12 of 54 patients (22.2%). CONCLUSION: CAPA can frequently manifest as COVID-19 pneumonia without common CT abnormalities of pulmonary aspergillosis. If abnormalities exist on CT images, CAPA may frequently accompany bronchial abnormalities.