A Multiplex PCR and DNA-Sequencing Workflow on Serum for the Diagnosis and Species Identification for Invasive Aspergillosis and Mucormycosis.

There has been significant increase in the use of molecular tools for the diagnosis of invasive aspergillosis (IA) and mucormycosis. However, their range of detection may be too limited as species diversity and coinfections are increasing. Here, we aimed to evaluate a molecular workflow based on a new multiplex PCR assay detecting the whole Aspergillus genus and the Mucorales order followed by a species-specific PCR or a DNA-sequencing approach for IA and/or mucormycosis diagnosis and species identification on serum. Performances of the MycoGENIE Aspergillus spp./Mucorales spp. duplex PCR kit were analyzed on a broad range of fungal strains and on sera from high-risk patients prospectively over a 12-month period. The kit allowed the detection of nine Aspergillus species and 10 Mucorales (eight genera) strains assessed. No cross-reactions between the two targets were observed. Sera from 744 patients were prospectively analyzed, including 35 IA, 16 mucormycosis, and four coinfections. Sensitivity varies from 85.7% (18/21) in probable/proven IA to 28.6% (4/14) in COVID-19-associated pulmonary aspergillosis. PCR-positive samples corresponded to 21 A. fumigatus, one A. flavus, and one A. nidulans infections. All the disseminated mucormycosis were positive in serum (14/14), including the four Aspergillus coinfections, but sensitivity fell to 33.3% (2/6) in localized forms. DNA sequencing allowed Mucorales identification in serum in 15 patients. Remarkably, the most frequent species identified was Rhizomucor pusillus (eight cases), whereas it is barely found in fungal culture. This molecular workflow is a promising approach to improve IA and mucormycosis diagnosis and epidemiology.

Revised workflow practices in the management of acute invasive fungal sinusitis during the coronavirus disease 2019 pandemic.

OBJECTIVE: To document changes in evaluation protocols for acute invasive fungal sinusitis during the coronavirus disease 2019 pandemic, and to analyse concordance between clinical and histopathological diagnoses based on new practice guidelines. METHODS: Protocols for the evaluation of patients with suspected acute invasive fungal sinusitis both prior and during the coronavirus disease 2019 period are described. A retrospective analysis of patients presenting with suspected acute invasive fungal sinusitis from 1 May to 30 June 2021 was conducted, with assessment of the concordance between clinical and final diagnoses. RESULTS: Among 171 patients with high clinical suspicion, 160 (93.6 per cent) had a final histopathological diagnosis of invasive fungal sinusitis, concordant with the clinical diagnosis, with sensitivity of 100 per cent, positive predictive value of 93.6 per cent and negative predictive value of 100 per cent. CONCLUSION: The study highlights a valuable screening tool with good accuracy, involving emphasis on 'red flag' signs in high-risk populations. This could be valuable in situations demanding the avoidance of aerosol-generating procedures and in resource-limited settings facilitating early referral to higher level care centres.

Invasive fungal infections in critically ill COVID-19 patients in a large tertiary university hospital in Israel.

An increasing number of studies have tried to determine the incidence of invasive fungal infections (IFIs) in COVID-19 patients. Challenges in the diagnosis of pulmonary aspergillosis in these patients have led to new definitions of COVID-19-associated pulmonary aspergillosis (CAPA). The aim of this study was to determine the incidence and outcomes of and risk factors for IFIs in critically-ill COVID-19 patients, using the new definitions, in a tertiary center in Israel. METHODS: A case-controlled study (from 1 September 2020 to 31 March 2021) in which data from COVID-19 critically-ill patients with a diagnosis of IFI were collected and compared to a control group without IFI. RESULTS: The incidence of IFI amongst 311 COVID-19 critically-ill patients was 6.1%. 3.5% had CAPA and 3.5% had candidemia. In-hospital mortality was higher amongst patients with IFI compared to those without IFI (89.4% vs 60%, p < 0.03). The most significant predictors of IFI were cardiovascular co-morbidity and carbapenem use. CONCLUSIONS: The low incidence of CAPA in our group of COVID-19 critically-ill patients was consistent with recent reports, underscoring the importance of differentiating between true infection and colonization. Awareness and timely diagnosis of IFIs in COVID-19 critically-ill patients are imperative considering the associated high mortality.

Post coronavirus disease mucormycosis: a deadly addition to the pandemic spectrum.

OBJECTIVE: To study the possible association between invasive fungal sinusitis (mucormycosis) and coronavirus disease. METHODS: A prospective observational study was conducted at a tertiary care centre over four months, involving all patients with mucormycosis of the paranasal sinuses suffering from or having a history of coronavirus disease infection. RESULTS: Twenty-three patients presented with mucormycosis, all had an association with coronavirus disease 2019. The ethmoids (100 per cent) were the most common sinuses affected. Intra-orbital extension was seen in 43.47 per cent of cases, while intracranial extension was only seen in 8.69 per cent. Diabetes mellitus was present in 21 of 23 cases, and was uncontrolled in 12 cases. All patients had a history of steroid use during their coronavirus treatment. CONCLUSION: New manifestations of coronavirus disease 2019 are appearing over time. The association between coronavirus and mucormycosis of the paranasal sinuses must be given serious consideration. Uncontrolled diabetes and over-zealous use of steroids are two main factors aggravating the illness, and both of these must be properly checked.

Diagnosis of invasive fungal disease in coronavirus disease 2019: approaches and pitfalls.

PURPOSE OF REVIEW: This review will comment on the current knowledge for the diagnosis of the main causes of COVID-19-associated invasive fungal disease (IFD); it will discuss the optimal strategies and limitations and wherever available, will describe international recommendations. RECENT FINDINGS: A range of secondary IFDs complicating COVID-19 infection have been described and while COVID-19-associated pulmonary aspergillosis was predicted, the presentation of significant numbers of COVID-19-associated candidosis and COVID-19-associated mucormycosis was somewhat unexpected. Given the range of IFDs and prolonged duration of risk, diagnostic strategies need to involve multiple tests for detecting and differentiating various causes of IFD. Although performance data for a range of tests to diagnose COVID-19-associated pulmonary aspergillosis is emerging, the performance of tests to diagnose other IFD is unknown or based on pre-COVID performance data. SUMMARY: Because of the vast numbers of COVID-19 infections, IFD in COVID-19 critical-care patients represents a significant burden of disease, even if incidences are less than 5%. Optimal diagnosis of COVID-19-associated IFD requires a strategic approach. The pandemic has highlighted the potential impact of IFD outside of the typical high-risk clinical cohorts, given the ever-increasing population at risk of IFD and enhanced surveillance of fungal infections is required.

A case report of rhino-facial mucormycosis in a non-diabetic patient with COVID-19: a systematic review of literature and current update.

BACKGROUND: COVID-19 disease may be associated with a wide range of bacterial and fungal infections. We report a patient with COVID-19 infection who developed rhino-facial mucormycosis during treatment with corticosteroids. CASE PRESENTATION: A 59-year-old non-diabetic male patient was admitted with a diagnosis of COVID-19 based on positive RT-PCR and CT of the lungs. Due to sever lung involvement, he was treated with methylprednisolone. The patient was re-admitted to hospital, due to nasal obstruction and left side facial and orbital swelling, several days after discharge. In sinus endoscopic surgery, debridement was performed and the specimens were sent to pathology and mycology laboratories. A nasal biopsy showed wide hyphae without septa. The sequenced PCR product revealed Rhizopus oryzae. Despite all medical and surgical treatment, the patient died. In addition, the characteristics of patients with COVID-19-associated mucormycosis were reviewed in 44 available literatures. In most studies, diabetes mellitus was the most common predisposing factor for mucormycosis. CONCLUSION: Our report highlights the need for assessing the presence of mucormycosis in patients with COVID-19 and also it shows that physicians should consider the potential for secondary invasive fungal infections in COVID-19 cases.

Invasive fungal infections among critically ill adult COVID-19 patients: First experiences from the national centre in Hungary.

INTRODUCTION: Data suggests that invasive fungal infections (IFI) might complicate COVID-19. Our goal was to describe characteristics of IFI among critically ill COVID-19 adults. METHODS: A retrospective observational case-series analysis was done between March-July 2020. Consecutive patients with critical COVID-19 were eligible, and have been included when proven or putative/probable IFI could be confirmed during their course. For COVID-19 diagnosis, ECDC definitions and WHO severity criteria were followed. Candidaemia was diagnosed according to the ESCMID 2012 guideline. Invasive pulmonary aspergillosis (IPA) was defined following EORTC/MSG, ECMM/ISHAM and modified AspICU criteria. Outcome variables were rates of IFIs, in-hospital all-cause mortality, rate and time to negative respiratory SARS-CoV-2 PCR. RESULTS: From 90 eligible patients, 20 (22.2%) fulfilled criteria for IFI. Incidence rate for IFI was 2.02 per 100 patient-days at ICU. Patients were mostly elderly males with significant comorbidities, requiring mechanical ventilation because of ARDS. IFI could be classified as candidaemia in 7/20 (40%), putative/probable IPA in 16/20 (80.0%). Isolated species of candidaemia episodes were Candida albicans (4/9, 44.4%), Candida glabrata (3/9, 33.3%), Candida parapsilosis (1/9, 11.1%), Candida metapsilosis (1/9, 11.1%). Mold isolates from lower respiratory tract were Aspergillus fumigatus, BAL galactomannan positivity was prevalent (16/20, 80.0%). Mortality was 12/20 (60.0%) with a median time to death of 31.0±37.0 (5-89) days. Only 9/20 (45.0%) patients reached SARS-CoV-2 PCR negativity after a median time of 20.0±12.0 (3-38) days. CONCLUSION: In this small cohort of critically ill COVID-19 adults, morbidity and mortality related to invasive fungal infections proved to be significant.

Aspergillus Polymerase Chain Reaction-An Update on Technical Recommendations, Clinical Applications, and Justification for Inclusion in the Second Revision of the EORTC/MSGERC Definitions of Invasive Fungal Disease.

Aspergillus polymerase chain reaction testing of blood and respiratory samples has recently been included in the second revision of the EORTC/MSGERC definitions for classifying invasive fungal disease. This is a result of considerable efforts to standardize methodology, the availability of commercial assays and external quality control programs, and additional clinical validation. This supporting article provides both clinical and technical justifications for its inclusion while also summarizing recent advances and likely future developments in the molecular diagnosis of invasive aspergillosis.

Evaluation of the Performance of the Associates of Cape Cod STAT Assay for the Diagnosis of Invasive Fungal Disease in Critical-Care Patients with COVID-19.

During the COVID-19 pandemic, there have been increasing reports of invasive fungal disease (IFD) in critical care, where rapid access to (1-3)-ß-d-glucan (BDG) testing may have enhanced diagnosis. The potential benefit of rapidly accessible BDG results is limited by local availability of BDG testing, with low demand resulting in testing being performed in specialist centers. The recent release of the Associates of Cape Cod STAT assay provides a simple, low-throughput BDG platform, potentially increasing accessibility. During the pandemic, BDG testing using the Fungitell assay (FA) was a critical component of screening for IFD in our critical care. The performance of the STAT was retrospectively determined through a case-control study of 107 serum samples from critical-care COVID-19 patients with IFD defined according to international guidelines. The STAT demonstrated excellent qualitative (observed agreement, 97.2%; kappa, 0.94) and quantitative (Spearman's coefficient, 0.8962) agreement with the FA. Sample positivity was greater (P < 0.0001) in samples from cases (67.7%) versus controls (6.1%). Using the manufacturer's threshold (≥1.2), sensitivity and specificity for the detection of proven/probable IFD were 67.9% and 93.9%, respectively. Using a lower positivity threshold of ≥0.87 increased sensitivity to 71.4% without compromising specificity. When the STAT BDG index was >2.86, specificity was 100%. The STAT provides a simple, comparable alternative to the FA for detecting BDG. Sensitivity is moderate, and specificity is excellent for the diagnosis of IFD in the critical-care COVID-19 patient. The potential for enhancing access to BDG testing through the uptake of STAT at centers where FA is not available is beneficial, especially during the COVID-19 pandemic.

Emerging Fungal Infections.

Various uncommon fungal pathogens have been increasingly identified as causes of disseminated and invasive fungal disease (IFD) worldwide. Growing recognition and clinical knowledge of these emerging fungal pathogens has occurred through improved molecular diagnostics, nucleic sequence databases, and taxonomic reclassification of medically significant fungi. However, emerging fungal diseases carry significant morbidity and mortality and, due to a paucity of published literature, the collective clinical experience with these fungi is often limited. In this review, we focus on unusual emerging fungal pathogens not extensively covered elsewhere in this issue of Infectious Diseases Clinics of North America.

[Invasive fungal infections in ICU patients - What's New?] / Invasive Pilzinfektionen bei Intensivpatienten ­ Was gibt es Neues?

Invasive fungal infections are gaining increasing importance in intensive care medicine. The aim of this article is to present an update on recent developments in the field of invasive fungal infection in critically ill patients. Particular emphasis is placed on the recently described invasive mold infections in patients with acute respiratory distress syndrome due to influenza or COVID-19. Detecting high-risk patients and the optimal diagnostic and therapeutic strategies play a decisive role to improve outcome.

Occurrence of Invasive Pulmonary Fungal Infections in Patients with Severe COVID-19 Admitted to the ICU.

Rationale: Whether severe coronavirus disease (COVID-19) is a significant risk factor for the development of invasive fungal superinfections is of great medical interest and remains, for now, an open question.Objectives: We aim to assess the occurrence of invasive fungal respiratory superinfections in patients with severe COVID-19.Methods: We conducted the study on patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related pneumonia admitted to five ICUs in France who had respiratory and serum sampling performed for specific screening of fungal complications.Measurements and Main Results: The study population included a total of 145 patients; the median age was 55 years old. Most of them were male (n = 104; 72%), were overweight (n = 99; 68%), and had hypertension (n = 83; 57%) and diabetes (n = 46; 32%). Few patients presented preexisting host risk factors for invasive fungal infection (n = 20; 14%). Their global severity was high; all patients were on invasive mechanical ventilation, and half (n = 73, 54%) were on extracorporeal membrane oxygenation support. Mycological analysis included 2,815 mycological tests (culture, galactomannan, ß-glucan, and PCR) performed on 475 respiratory samples and 532 sera. A probable/putative invasive pulmonary mold infection was diagnosed in 7 (4.8%) patients and linked to high mortality. Multivariate analysis indicates a significantly higher risk for solid organ transplant recipients (odds ratio, = 4.66; interquartile range, 1.98-7.34; P = 0.004). False-positive fungal test and clinically irrelevant colonization, which did not require the initiation of antifungal treatment, was observed in 25 patients (17.2%).Conclusions: In patients with no underlying immunosuppression, severe SARS-CoV-2-related pneumonia seems at low risk of invasive fungal secondary infection, especially aspergillosis.

Systemic mycoses: a potential alert for complications in COVID-19 patients.

As the global COVID-19 pandemic spreads worldwide, new challenges arise in the clinical landscape. The need for reliable diagnostic methods, treatments and vaccines for COVID-19 is the major worldwide urgency. While these goals are especially important, the growing risk of co-infections is a major threat not only to the health systems but also to patients' lives. Although there is still not enough published statistical data, co-infections in COVID-19 patients found that a significant number of patients hospitalized with COVID-19 developed secondary systemic mycoses that led to serious complications and even death. This review will discuss some of these important findings with the major aim to warn the population about the high risk of concomitant systemic mycoses in individuals weakened by COVID-19.