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.

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.

Central Retinal Artery Occlusion in Rhino-Orbital-Cerebral Mucormycosis: An Inflammatory-Prothrombotic State.

PURPOSE: The aim was to evaluate patient profiles of rhino-orbital-cerebral mucormycosis (ROCM) cases with central retinal artery occlusion (CRAO) postcoronavirus disease 2019. DESIGN: A nonrandomized retrospective case-control study. METHODS: The ROCM cases presenting with CRAO were compared with a control ROCM group without CRAO at a tertiary care center. Demography, systemic status, clinical features, histopathology, imaging, and blood profile were assessed for any specific risk factors. RESULTS: A total of 12 patients were seen in the CRAO group and 16 in the non-CRAO group. The male-to-female ratio was 3:1 with a mean age of 49.5 years. In the CRAO group, 75% had diabetes mellitus with mean hemoglobin A1c of 9.03%, and 66.7% had received steroid treatment. All cases were histopathologically confirmed positive for mucor. There was a significant difference in mean D-dimer and serum ferritin between the 2 groups, with higher level in the CRAO group. All patients with CRAO had light perception-negative vision, with total ophthalmoplegia and proptosis seen in 66.7% of cases. Four patients had orbital apex involvement, 5 had cavernous sinus involvement, and 8 had intracranial involvement in the CRAO group. CONCLUSIONS: Inflammatory markers D-dimer and serum ferritin were significantly associated with CRAO, suggestive of hyperinflammatory and hypercoagulable state. A high index of suspicion should be maintained in cases with elevated markers and prophylactic anticoagulants can be started to prevent CRAO in a subset of patients.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Disseminated pulmonary mucormycosis with concomitant tuberculosis infection in a diabetic patient.

Patients with diabetes are often susceptible to various opportunistic infections such as tuberculosis and mucormycosis. However, the occurrence of both these infections simultaneously is rare. We present one such case of pulmonary tuberculosis with disseminated pulmonary mucormycosis in a patient with diabetes, which was successfully managed.

Mucorales DNA detection in serum specimens for early diagnosis of mucormycosis.

We report a case of pulmonary mucormycosis in a patient with T-cell acute lymphoblastic leukemia. The diagnosis of mucormycosis was initially based on mycological examination of a pulmonary specimen. However, we describe how it could have been made 2 months earlier using polymerase chain reaction assays targeting Mucorales species on serum specimens.

Detection of Rhizopus-specific antigen in human and murine serum and bronchoalveolar lavage.

Mucormycosis is a deep-seated fungal infection that mainly develops in patients with severe immunodeficiencies such as those with malignant hematological diseases. Despite poor prognosis, there is no reliable and minimally invasive diagnostic method-such as serodiagnosis-for making a clinical decision regarding the condition. As early diagnosis and early treatment improve the prognosis of mucormycosis, the development of a sensitive early diagnostic method is important. We had previously identified a Rhizopus-specific antigen (RSA) by signal sequence trapping and retrovirus-mediated expression (SST-REX), and evaluated its utility as a diagnostic antigen by constructing a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect serum RSA levels in inoculated mice. In this study, we used the RSA-specific rabbit monoclonal antibodies generated by novel hybridoma technology to improve the sensitivity of the ELISA system. We observed an increase in serum and bronchoalveolar lavage fluid (BALF) levels of RSA in mouse model 1 day after inoculation, suggesting that this newly developed monoclonal antibody-based ELISA system may be useful for the diagnosis of mucormycosis in the early stages of infection. In addition, we measured RSA levels in human serum and BALF, and found that serum RSA level was higher in mucormycosis patients (15.1 ng/ml) than that in invasive pulmonary aspergillosis patients (0.53 ng/ml) and the negative control (0.49 ng/ml). Our results suggest that RSA may be a powerful tool for the diagnosis of pulmonary mucormycosis, and its differentiation from other deep-seated mycoses such as aspergillosis.

Sporulation on blood serum increases the virulence of Mucor circinelloides.

Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1ß, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.

Matched-paired analysis of patients treated for invasive mucormycosis: standard treatment versus posaconazole new formulations (MoveOn).

BACKGROUND: First-line antifungal treatment for invasive mucormycosis (IM) consists of liposomal amphotericin B. Salvage treatment options are limited and often based on posaconazole oral suspension. With the approval of posaconazole new formulations, patients could benefit from improved pharmacokinetics, safety and tolerability. OBJECTIVES: Our aim was to assess the effectiveness of posaconazole new formulations for IM treatment. METHODS: We performed a case-matched analysis with proven or probable IM patients from the FungiScope® Registry. First-line posaconazole new formulations (1st-POSnew) and first-line amphotericin B plus posaconazole new formulations (1st-AMB+POSnew) cases were matched with first-line amphotericin B-based (1st-AMB) treatment controls. Salvage posaconazole new formulations (SAL-POSnew) cases were matched with salvage posaconazole oral suspension (SAL-POSsusp) controls. Each case was matched with up to three controls (based on severity, haematological/oncological malignancy, surgery and/or renal dysfunction). RESULTS: Five patients receiving 1st-POSnew, 18 receiving 1st-AMB+POSnew and 22 receiving SAL-POSnew were identified. By day 42, a favourable response was reported for 80.0% (n = 4/5) of patients receiving 1st-POSnew, for 27.8% (n = 5/18) receiving 1st-AMB+POSnew and for 50.0% (n = 11/22) receiving SAL-POSnew. Day 42 all-cause mortality of patients receiving posaconazole new formulations was lower compared with controls [20.0% (n = 1/5) in 1st-POSnew versus 53.3% (n = 8/15) in 1st-AMB; 33.3% (n = 6/18) in 1st-AMB+POSnew versus 52.0% (n = 26/50) in 1st-AMB; and 0.0% (n = 0/22) in SAL-POSnew versus 4.4% (n = 2/45) in SAL-POSsusp]. CONCLUSIONS: Posaconazole new formulations were effective in terms of treatment response and associated mortality of IM. While posaconazole new formulations may be an alternative for treatment of IM, the limited sample size of our study calls for a cautious interpretation of these observations.

Diagnostic performance of immunohistochemistry for the aspergillosis and mucormycosis.

OBJECTIVE: To investigate the accuracy of immunohistochemistry (IHC) tests for distinguishing between mucormycosis and aspergillosis and compare the clinical characteristics of mucormycosis patients according to galactomannan (GM) results. METHODS: We evaluated diagnostic performance of IHC test with tissue sections of patients with culture-proven invasive fungal infection. In addition, we conducted PCR assay with tissue sections of mucormycosis patients with positive GM results to evaluate the possibility of co-infection. RESULTS: In culture-proven mucormycosis (n = 13) and aspergillosis (n = 20), the sensitivity and specificity of IHC test were both 100% for mucormycosis and 85% and 100%, respectively, for aspergillosis. Among the 53 patients who met the modified criteria for proven mucormycosis and had GM assay results, 24 (45%) were positive. Compared with those with negative GM results (n = 29), mucormycosis patients with positive GM results had significantly higher incidence of gastrointestinal tract infections (6/24 [25%] vs 0/29 [0%], P = .006) and were more likely to be histomorphologically diagnosed as aspergillosis (7/24 [29%] vs 2/29 [7%], P = .06). PCR assay amplified both Aspergillus- and Mucorales-specific DNA in 6 of these 24 cases. CONCLUSIONS: Immunohistochemistry tests seem useful for compensating for the limitations of histomorphologic diagnosis in distinguishing between mucormycosis and aspergillosis. Some proven mucormycosis patients with positive GM results had histopathology consistent with aspergillosis and gastrointestinal mucormycosis. In addition, about one quarter of these patients revealed the evidence of co-infection with aspergillosis by PCR assay.

Mucormycosis in South America: A review of 143 reported cases.

Mucormycosis is a rare but important invasive fungal disease that most often affects immunocompromised hosts. The incidence of mucormycosis appears to be increasing worldwide, as risk factors such as the use of immunosuppressive therapies become more common. We report the results of a literature review of 143 mucormycosis cases reported in South America between 1960 and 2018. The number of reported cases has increased by decade, from 6 in the 1960s to 51 in the 2010s. The most common underlying conditions associated with mucormycosis in South America were diabetes mellitus (42.0%) and penetrating trauma/burns (20.0%). Underlying conditions involving immunosuppression, including treatment of haematologic malignancy, solid organ transplant, and corticosteroid use, also accounted for a large proportion of cases (45.5%). Between 1960 and 2018, cases of mucormycosis associated with conditions involving immunosuppression accounted for the highest mortality rate (58.5%), followed by diabetes mellitus (45.0%), and penetrating trauma/burns (37.9%). Overall mortality decreased from 100% to 39.4% during this period, mainly driven by the increasing availability and use of antifungal therapies and surgical intervention. However, these treatments are not yet universally utilised across the region in the treatment of mucormycosis; efforts to improve availability of effective treatments would be likely to improve outcomes.

Defining breakthrough invasive fungal infection-Position paper of the mycoses study group education and research consortium and the European Confederation of Medical Mycology.

Breakthrough invasive fungal infections (IFIs) have emerged as a significant problem in patients receiving systemic antifungals; however, consensus criteria for defining breakthrough IFI are missing. This position paper establishes broadly applicable definitions of breakthrough IFI for clinical research. Representatives of the Mycoses Study Group Education and Research Consortium (MSG-ERC) and the European Confederation of Medical Mycology (ECMM) reviewed the relevant English literature for definitions applied and published through 2018. A draft proposal for definitions was developed and circulated to all members of the two organisations for comment and suggestions. The authors addressed comments received and circulated the updated document for approval. Breakthrough IFI was defined as any IFI occurring during exposure to an antifungal drug, including fungi outside the spectrum of activity of an antifungal. The time of breakthrough IFI was defined as the first attributable clinical sign or symptom, mycological finding or radiological feature. The period defining breakthrough IFI depends on pharmacokinetic properties and extends at least until one dosing interval after drug discontinuation. Persistent IFI describes IFI that is unchanged/stable since treatment initiation with ongoing need for antifungal therapy. It is distinct from refractory IFI, defined as progression of disease and therefore similar to non-response to treatment. Relapsed IFI occurs after treatment and is caused by the same pathogen at the same site, although dissemination can occur. These proposed definitions are intended to support the design of future clinical trials and epidemiological research in clinical mycology, with the ultimate goal of increasing the comparability of clinical trial results.

Correlation of fungal cultures from non-sterile sites and Galactomannan assay with the diagnosis of aspergillosis and mucormycosis based on sterile culture results and histopathologic findings.

BACKGROUND: Data on whether positive non-sterile fungal culture has the same clinical value as a positive galactomannan (GM) result are limited. METHODS: Patients with biopsy-proven invasive aspergillosis or mucormycosis (over an 8-year period) in whom the results of GM and fungal culture of sputum and/or sinus aspirates were available were enrolled. Biopsy-proven cases were defined if fungal culture from a sterile biopsy specimen gave a positive result and/or hyphae were demonstrated by immunohistochemical staining for aspergillosis and mucormycosis. RESULTS: A total of 71 patients comprising 30 biopsy-proven cases of aspergillosis including 13 cases with positive sterile cultures and 41 biopsy-proven cases of mucormycosis including eight cases with positive sterile cultures were enrolled. Of 30 patients with aspergillosis, 15 (50%) revealed Aspergillus spp. growth from non-sterile site and none exhibited the agents of mucormycosis growth from non-sterile site. However, of 41 patients with mucormycosis, eight (20%) revealed the agents of mucormycosis growth from non-sterile site and three (7%) exhibited Aspergillus spp. growth from non-sterile site. In terms of GM assays, 23 (77%) of 30 patients with aspergillosis revealed positive GM results, and 17 (41%) of 41 patients with mucormycosis revealed positive GM assays. So, positive fungal culture from non-sterile site (88% [23/26]) were better correlated with the diagnosis than positive GM assay (57% [23/40]) (p value = .01). CONCLUSIONS: Positive fungal cultures from non-sterile sites better correlate with the diagnosis of aspergillosis and mucormycosis based on sterile culture results and histopathological findings than positive GM results.

Invasive hepatic mucormycosis: A case report and review of the literature.

Mucormycosis generally develops under immunocompromised conditions, including hematological malignancies and solid organ or hematopoietic stem cell transplantation. Although mucormycosis usually affects the lungs and paranasal sinuses, sporadic cases of invasive mucormycosis of the liver have been reported. We hereby report a patient with myelofibrosis who developed hepatic mucormycosis diagnosed by post-mortem examination. An extensive literature review identified 13 reported cases of hepatic mucormycosis, including ours, without lung involvement. Most of the underlying diseases or conditions were hematological malignancies and solid organ transplantation. Three cases had splenic lesions and four had gastrointestinal lesions, suggesting the possibility of translocation to the liver and/or spleen from the gastrointestinal tracts. Hepatic mucormycosis should be recognized as one of the presentations of invasive mucormycosis, especially when hepatic nodules are found in immunocompromised patients such as those with hematological malignancy or recipients of solid organ transplantation.

Pharmacokinetics of Intravenous Isavuconazole in Solid-Organ Transplant Recipients.

Isavuconazole may be useful in treating and preventing fungal infections in solid-organ transplant (SOT) recipients due to its safety profile and activity against Aspergillus and some Mucorales Isavuconazole has favorable pharmacokinetics based on clinical trials in various patient populations, but data are limited in SOT recipients. We evaluated the steady-state pharmacokinetics of isavuconazole in 26 SOT recipients receiving the drug intravenously for prophylaxis. There was moderate interpatient variability in isavuconazole pharmacokinetic parameters (coefficients of variation of 51% for the area under the plasma concentration-versus-time curve [AUC] and 59% for the trough plasma concentration [Ctrough]). AUC and steady-state Ctrough were significantly lower in women, patients with a body mass index of ≥18.5 kg/m2, and those receiving hemodialysis. Trough plasma concentrations were highly correlated with AUCs (R2 = 0.94) and can serve as a suitable measure of isavuconazole exposure in patients. In conclusion, moderate interpatient variability in isavuconazole exposure, the identification of factors associated with lower exposure, the recognition that Ctrough is a surrogate marker for AUC, and the availability of a simple analytical method suggest that therapeutic drug monitoring (TDM) may be useful for guiding treatment in at least some SOT recipients. Future studies are needed to correlate isavuconazole exposure with patients' clinical outcomes and to determine the clinical role of TDM.

Invasive cutaneous mucormycosis in a preterm neonate presenting as a vesicobullous lesion.

Mucormycosis is a relatively rare fungal infection seen in immunocompromised patients. Very few cases of invasive cutaneous mucormycosis occurring in neonates have been reported in literature. It is an aggressive disease with a mortality rate of around 64% in neonates, so a high index of suspicion is essential for rapid diagnosis and definitive treatment with broad-spectrum antifungals such as Amphotericin B. We present a case of a premature infant born at 25 weeks of gestation who developed vesicobullous lesions all over the body on day 5 of life. Biopsy from the vesicles confirmed the presence of angioinvasive fungal hyphae of mucormycosis which were highlighted on Periodic acid-Schiff and Grocott stain.

Disseminated mucormycosis masquerading as rejection early after orthotopic heart transplantation.

Mucorales organisms are an uncommon cause of invasive fungal infections after solid organ transplantation but are associated with great morbidity and mortality. We report a fatal case of disseminated Cunninghamella infection early after heart transplantation. The patient developed graft dysfunction and elevated markers of myocyte injury and autopsy revealed fulminant fungal myocarditis. This case highlights the need for a high index of suspicion in immunocompromised patients who are not improving with standard antimicrobial therapy.

Clinical implications of serum galactomannan measurement in patients with acute invasive fungal rhinosinusitis.

BACKGROUND: Acute invasive fungal rhinosinusitis (AIFR) is an aggressive opportunistic infection with a high mortality rate. Recently, non-invasive techniques have been introduced for diagnosis of invasive fungal disease. The purpose of this study is to evaluate the diagnostic significance of serum galactomannan measurement in patients with AIFR. METHODOLOGY: We conducted a retrospective case-control study of 28 patients with AIFR and 36 fungus ball (FB) patients. We evaluated clinical, laboratory, and pathologic findings along with disease course. RESULTS: In 28 patients with AIFR, there were 21 cases of invasive aspergillosis (IA) and 7 cases of invasive mucormycosis (IM). The control group was comprised of 36 patients with FB. The three-group analysis showed a statistically significant difference among the groups. At the cut-off value of 0.48, the sensitivity and specificity were 71.4% and 93.0%, respectively. Comparison of mean serum galactomannan levels in 5 non-survivors and 9 survivors at initial measurement showed no significant difference, but that became significantly different 1 week later. Statistical analysis showed that the levels of serum galactomannan decreased significantly according to the measurement-point in within survivor-group analysis. The difference in between survivor-groups analysis was also significant. CONCLUSION: Serum galactomannan measurement seems useful for early diagnosis and discrimination of fungal species in patients with AIFR. In addition, clinical outcomes may be related to the levels and patterns of serum galactomannan, especially in IA. The appropriate measurement of galactomannan might be helpful in treating the patients at high risk for AIFR.