A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Clinical Profile of Mucormycosis Patients during Coronavirus Disease 2019 Pandemic: A Retrospective Observational Study.

BACKGROUND: During the coronavirus disease (COVID-19) pandemic, an increased incidence of mucormycosis infection was noted globally, the majority being from India. We aimed to study the clinical profile of the mucormycosis patients during the COVID-19 pandemic admitted at tertiary care centers. MATERIALS AND METHODS: This is a retrospective record-based observation study conducted at Gandhi Medical College, Bhopal. All suspected or laboratory-proven mucormycosis patients were included. Detailed data on demography, clinical features, risk factors, laboratory/radiological findings, and outcomes were recorded. RESULTS: A total of 288 patients were enrolled and 121(42%) showed mucormycosis on potassium hydroxide (KOH) mount. The mean age was 51.52 ± 10.88 years, male:female ratio was 2.3:1. Most common symptom was facial swelling/pain and fever. The most common risk factor was COVID-19 infection (78.5%) followed by the presence of diabetes mellitus (DM) (70.8%) out of which 152 (52.8%) patients were previously diagnosed cases and 52 (18%) patients were newly diagnosed, 159 (55.2%) had a history of corticosteroid use, 87 (30.2%) had a history of use of oxygen support and 67 (23.2%) had hypertension. Most patients had invasion limited to sinus (46.5%) but the presence of DM was associated with an increased risk of cerebral invasion. Out of 288 patients admitted with mucormycosis, 31 patients collapsed to death while the remaining 257 patients were discharged from the hospital. CONCLUSION: It is observed that during the COVID-19 pandemic, hyperglycemia and inappropriate use of corticosteroids were associated with an increased risk of development of mucormycosis in patients with or without DM. We conclude that regular blood glucose monitoring, adequate glycemic control, and judicious evidence-based use of corticosteroids and immunosuppressants in COVID-19 are recommended to reduce the emergence of mucormycosis in such circumstances.

Mucormycosis in Liver Allograft Following Transplant for Secondary Biliary Cirrhosis.

Mucormycosis, a group of opportunistic mycoses caused by Mucorales, present a significant threat to immunocompromised patients. In this report, we present the case of a 57-year-old male patient who underwent liver transplant for secondary biliary cirrhosis following inadvertent bile duct injury. Despite initial satisfactory postoperative evolution, the patient developed fever, and imaging revealed a suspicious lesion. Preliminary culture growth suggested a filamentous fungus, leading to initiation of liposomal amphotericin B. However, the lesion progressed, and a surgical debridement was necessary. During surgery, involvement of the liver dome and diaphragm was observed, and a nonanatomical hepatectomy was performed. Despite efforts, the patient's condition deteriorated, ultimately resulting in multiple organ failure and mortality. This case emphasizes the challenging nature of mucormycosis in livertransplant recipients.

Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

Aspergillus species and Mucorales agents are the primary etiologies of invasive fungal disease (IFD). Biomarkers that predict outcomes are needed to improve care. Patients diagnosed with invasive aspergillosis and mucormycosis using plasma cell-free DNA (cfDNA) PCR were retested weekly for 4 weeks. The primary outcome included all-cause mortality at 6 weeks and 6 months based on baseline cycle threshold (CT) values and results of follow-up cfDNA PCR testing. Forty-five patients with Aspergillus and 30 with invasive Mucorales infection were retested weekly for a total of 197 tests. Using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) criteria, 30.7% (23/75), 25.3% (19/75), and 38.7% (29/75) had proven, probable, and possible IFD, respectively. In addition, 97.3% (73/75) were immunocompromised. Baseline CT increased significantly starting at week 1 for Mucorales and week 2 for Aspergillus. Aspergillosis and mucormycosis patients with higher baseline CT (CT >40 and >35, respectively) had a nonsignificantly higher survival rate at 6 weeks, compared with patients with lower baseline CT. Mucormycosis patients with higher baseline CT had a significantly higher survival rate at 6 months. Mucormycosis, but not aspergillosis patients, with repeat positive cfDNA PCR results had a nonsignificantly lower survival rate at 6 weeks and 6 months compared with patients who reverted to negative. Aspergillosis patients with baseline serum Aspergillus galactomannan index <0.5 and <1.0 had significantly higher survival rates at 6 weeks when compared with those with index ≥0.5 and ≥1.0, respectively. Baseline plasma cfDNA PCR CT can potentially be used to prognosticate survival in patients with invasive Aspergillus and Mucorales infections. IMPORTANCE: We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.

[Mucormycosis in the time of COVID-19: risks and challenges]. / Die Mukormykose in Zeiten von COVID-19: Risiken und Herausforderungen.

The first patients positive for SARS-CoV-2 were registered in December 2019. In March 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a global pandemic, the beginning of a worldwide health crisis that revealed numerous medical challenges for healthcare systems and pandemic emergency strategies.Among these challenges, mucormycosis, a typically rare fungal infection, gained global attention. With an average global incidence of about 2 per 1 million people, mucormycosis is considered a very rare disease, an opportunistic infection mostly affecting the lungs or skin and soft tissues in immunocompromised patients. Poorly controlled diabetes mellitus is one of the leading risk factors for rhino-orbital mucormycosis. Countries with a high prevalence of diabetes and limited healthcare resources have higher mucormycosis rates, with India and Pakistan being among the nations with particularly high incidences.During the second wave of the COVID-19 pandemic in India, mucormycosis rates surged dramatically within a few weeks, with over 47,500 cases of COVID-19-associated mucormycosis (CAM) reported between May and August 2021. Mucormycosis is characterized by a high mortality rate of up to 90%, especially when the diagnosis is delayed, and treatment commences late. There were concerns about a potentially global threat.In this article, we explore the risk factors and mechanisms leading to this viral-fungal coinfection. We present global distribution patterns, clinical presentation, and challenges in the diagnosis and treatment of COVID-19-associated mucormycosis.

Oral mucormycosis associated with COVID-19 and diabetes mellitus: Case report and literature review / Mucormicosis oral asociada a COVID-19 y diabetes mellitus: descripción de un caso.

Mucormycosis is an invasive opportunistic fungal infection with high mortality, mainly detected in people with COVID-19, especially those with underlying diseases such as diabetes mellitus. Mucormycosis prevalence is 0.005 to 1.7 cases per million inhabitants, and it has been increasing in countries like India and Pakistan. This mycosis can affect different organs, and clinical manifestations reflect the transmission mechanism. Frequent forms are rhino-orbital-cerebral and pulmonary. This disease should be suspected in patients with necrotic injuries on mucous membranes or skin. We present a case of a patient with diabetes mellitus and diagnosed with oral mucormycosis associated with COVID-19.

La mucormicosis es una infección fúngica oportunista e invasiva, con una elevada tasa de mortalidad. Se ha detectado principalmente en pacientes con COVID-19, especialmente en personas con enfermedades concomitantes como la diabetes mellitus. La prevalencia de las mucormicosis es de 0,005 a 1,7 casos por millón de habitantes y ha ido en aumento en países como India y Pakistán; puede afectar diferentes órganos y su forma clínica refleja el mecanismo de transmisión. Entre las formas frecuentes están la rino-orbital-cerebral y la pulmonar, por ello, debe sospecharse mucormicosis en los pacientes con lesiones necróticas en mucosas o piel. Se presenta el caso de un paciente con antecedentes de diabetes mellitus que fue diagnosticado con mucormicosis oral asociada a la COVID-19.

Investigations of an increased incidence of non-Aspergillus invasive mould infections in an onco-haematology unit.

AIMS OF THE STUDY: Invasive mould infections are life-threatening complications in patients with haematologic cancer and chemotherapy-induced neutropenia. While invasive aspergillosis represents the main cause of invasive mould infections, non-Aspergillus mould infections, such as mucormycosis, are increasingly reported. Consequently, their local epidemiology should be closely monitored. The aim of this study was to investigate the causes of an increased incidence of non-Aspergillus mould infections in the onco-haematology unit of a Swiss tertiary care hospital. METHODS: All cases of proven and probable invasive mould infections were retrospectively identified via a local registry for the period 2007-2021 and their incidence was calculated per 10,000 patient-days per year. The relative proportion of invasive aspergillosis and non-Aspergillus mould infections was assessed. Factors that may affect invasive mould infections' incidence, such as antifungal drug consumption, environmental contamination and changes in diagnostic approaches, were investigated. RESULTS: A significant increase of the incidence of non-Aspergillus mould infections (mainly mucormycosis) was observed from 2017 onwards (Mann and Kendall test p = 0.0053), peaking in 2020 (8.62 episodes per 10,000 patient-days). The incidence of invasive aspergillosis remained stable across the period of observation. The proportion of non-Aspergillus mould infections increased significantly from 2017 (33% vs 16.8% for the periods 2017-2021 and 2007-2016, respectively, p = 0.02). Building projects on the hospital site were identified as possible contributors of this increase in non-Aspergillus mould infections. However, novel diagnostic procedures may have improved their detection. CONCLUSIONS: We report a significant increase in non-Aspergillus mould infections, and mainly in mucormycosis infections, since 2017. There seems to be a multifactorial origin to this increase. Epidemiological trends of invasive mould infections should be carefully monitored in onco-haematology units in order to implement potential corrective measures.

Molecular mechanisms that govern infection and antifungal resistance in Mucorales.

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

Use of the Mucorales qPCR on blood to screen high-risk hematology patients is associated with better survival.

Our objective was to determine whether the twice-weekly screening of high-risk hematology patients by Mucorales qPCR on serum affects the prognosis of mucormycosis. Results from all serum Mucorales qPCR tests performed on patients from the hematology unit from January 2017 to December 2022 were analyzed. Patients with positive results were classified as having proven, probable or 'PCR-only' mucormycosis. One-month mortality for the local cohort was compared with that of a national cohort of cases of mucormycosis collected by the French surveillance network for invasive fungal disease ('Réseau de surveillances des infections fongiques invasives en France' (RESSIF)) from 2012 to 2018. From 2017 to 2022, 7825 serum Mucorales qPCR tests were performed for patients from the hematology unit; 107 patients with at least one positive Mucorales qPCR (164 positive samples) were identified. Sixty patients (70 positive samples, median Cq = 40) had no radiological criteria for mucormycosis and were considered not to have invasive fungal disease (70/7825, 0.9% false positives). It was not possible to classify disease status for six patients (12 positive samples, median Cq = 38). Forty-one patients (82 positive samples, median Cq = 35) had a final diagnosis of mucormycosis. In comparison with the RESSIF cohort, the local cohort was independently associated with a 48% lower one-month all-cause mortality rate (age-, sex-, and primary disease-adjusted hazard ratio = 0.52; 95% confidence interval: 0.29-0.94; P 0.03). Proactive screening for invasive mold diseases in high-risk hematology patients, including twice-weekly Mucorales qPCR on serum, was associated with mucormycosis higher survival.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Dermatopathologist accuracy in classifying angioinvasive fungal infections using virtual microscopy.

BACKGROUND: Due to perceived difficulty in the categorization of angioinvasive fungal infections based on histopathology, variation exists in dermatopathology reporting. METHODS: This study characterized the diagnosis of angioinvasive fungal infections by light microscopy at a single academic institution over an 11-year period. Subsequently, the accuracy of blinded reclassification by virtual microscopy was measured. RESULTS: Seventy-six specimens with hematoxylin-eosin slides were obtained from 33 patients. The mean diagnostic accuracy of dermatopathologists in differentiating mucormycosis, hyalohyphomycosis, and phaeohyphomycosis based on blinded reclassification via virtual microscopy was 74%, with a range of 65%-91%. CONCLUSIONS: While there was a range in diagnostic accuracy, the highest score of 91% and the identification of common sources of error suggest that histopathologic categorization of angioinvasive fungal infections can frequently be performed. However, accurate identification is not always possible given common pitfalls in diagnosis. In addition, standardized and clinically useful reporting should be considered.