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A clinicopathologic picture caused by Lichtheimia sp. in a 33-year-old male Sapajus xanthosternos kept in a conservationist center is described. Acute apathy, vomiting, fever, and polydipsia were associated with pneumonia, enteritis, and gastritis presenting fibrinoid vasculitis containing hyphae. The fungi were demonstrated molecularly, highlighting the spontaneous disease in primates., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

To estimate the diagnostic performance of Mucorales polymerase chain reaction (PCR) in Bronchoalveolar lavage fluid (BALF) in routine practice. This was a single-center retrospective study including all consecutive patients >18 years who underwent Mucorales PCR assay in BALF between January 2021 and May 2022. Index testing was prospectively performed using the MycoGENIE Aspergillus spp.-Mucorales spp. PCR. The reference was the diagnosis of pulmonary mucormycosis by the Adjudication Committee. Mucorales PCR in BALF was performed for 938 patients and was positive for 21 of 938 (2.2%). Eleven pulmonary mucormycosis (including one disseminated) were diagnosed. Among them, one (9.1%) was classified as proven mucormycosis, three (27.3%) as probable, and seven (63.6%) as possible according to the EORTC/MSGERC 2019 criteria. The main host factor was hematological malignancy (10 of 11, 90.9%). Mucorales PCR was positive in serum for eight patients (72.7%). Three patients had positive PCR in BALF, but negative in serum. The mean cycle threshold value was significantly lower in mucormycosis than false-positive cases. Sensitivity was 72.7% (95% confidence interval [CI], 43.4-90.3%), and specificity was 98.6% (95% CI, 97.6-99.2%). The positive and negative predictive values were 38.1% (95% CI, 20.8-59.1%) and 99.7% (95% CI, 99.1-99.9%), respectively. Mucorales PCR in BALF showed good diagnostic performance for mucormycosis, particularly in combination with serum PCR. A positive result should be interpreted with caution, given the possibility of carriage in the airway. However, its high negative predictive value and specificity suggest the utility of Mucorales PCR in BALF in the diagnosis of pulmonary mucormycosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175 bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The molecular composition and structural organization of the cell wall of filamentous fungi underlie the ability of the host to identify them as pathogens. Although the organization of the fungal cell wall, composed of 90% polysaccharides, is similar from one fungus to another, small variations condition their ability to trigger pattern recognition receptors. Because the incidence of mucormycosis, an emerging life-threatening infection caused by the species of the order Mucorales is increasing worldwide, the precise composition of the cell wall of two strains of Lichtheimia corymbifera was investigated in the early growth stages of germination (spores and germ-tubes) using trimethylsilylation and confocal microscopy. This study also characterizes the response of THP-1 cells to Mucorales. The study identified the presence of uncommon monosaccharides (fucose, galactose, and glucuronic acid) whose respective proportions vary according to the germination stage, revealing early parietal reorganization. Immunofluorescence studies confirmed the exposure of β-glucan on the surface of swollen spores and germ-tubes. Both spores and germ-tubes of L. corymbifera promoted an early and strong pro-inflammatory response, through TLR-2. Our results show the singularity of the cell wall of the order Mucorales, opening perspectives for the development of specific diagnostic biomarkers., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

PCR-based methods applied to various body fluids emerged in recent years as a promising approach for the diagnosis of mucormycosis. In this study, we set up and assess the value of a qPCR to detect a wide variety of Mucorales species in a single tube. A pair of degenerated primers targeting the rDNA operon was used in a qPCR utilizing an intercalating fluorescent dye. Analytical assessment, using a wide variety of both Mucorales strains (8 genera, 11 species) and non-Mucorales strains (9 genera, 14 species), showed 100% sensitivity and specificity rates with a limit of detection at 3 rDNA copy/qPCR reaction. Subsequently, 364 clinical specimens from 166 at-risk patients were prospectively tested with the assay. All the seven patients classified as proven/probable mucormycosis using the EORTC-MSG criteria had a positive qPCR as well as a patient with a proven uncharacterized invasive mold infection. In addition, three out of seven patients with possible mold invasive infections had at least one positive qPCR test. Sensitivity was calculated between 73.33 and 100% and specificity between 98.10 and 100%. The qPCR method proposed showed excellent performances and would be an important adjunctive tool for the difficult diagnosis of mucormycosis diagnosis., Lay Abstract: qPCR-based diagnosis is the most reliable approach for mucormycosis. We set up a pan-Mucorales qPCR able to detect in a single reaction not less than 11 different species. Both analytical and clinical performances support its use in the clinical setting., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Saksenaea erythrospora is a filamentous fungus belonging to the order Mucorales. Cases of cutaneous mucormycosis caused by Saksenaea spp. have previously been reported in immunocompetent and immunosuppressed people. A premature, 1-day-old bull calf from Texas with numerous plaque-like and ulcerative lesions in the skin was found at necropsy to have multiple areas of mycotic dermatitis and abomasitis. Fungal culture of the skin followed by morphological characterization and genetic analysis identified the etiologic agent as S. erythrospora.

The present case report describes the isolation of the fungus Cokeromyces recurvatus from the intestinal tract of a canine patient. Infection by this fungus is rare, having only been reported in 8 human beings and 1 cat. The fungus is not reported to exhibit tissue invasion. Cokeromyces recurvatus is most likely an opportunistic agent, and most cases described involve some degree of immunosuppression.

An adult male finless porpoise (Neophocaena phocaenoides) kept in an aquarium in Japan displayed loss of appetite and reduced body weight over several months. Necropsy examination revealed the presence of lesions in the pericardium, lung, and mediastinal and pancreatico-duodenal lymph nodes. Microscopically, these comprised regions of necrotizing granulomatous inflammation with multinucleated giant cells and surrounding fibrosis. Fungal hyphae were identified within macrophages and the extracellular tissue. Immunohistochemical labelling determined that these organisms were of the order Mucorales. A diagnosis of granulomatous pericarditis associated with systemic mucormycosis was made.

Cokeromyces recurvatus, a zygomycete, was isolated by fungal culture from the peritoneal fluid of a cat with jejunal perforation secondary to intestinal lymphosarcoma. This organism has not been recovered previously from a veterinary patient. The tissue form of C. recurvatus is morphologically similar to those of Coccidioides immitis and Paracoccidioides brasiliensis and may be misdiagnosed as 1 of these organisms on the basis of cytologic or histopathologic specimens, particularly in geographic regions where these organisms are not endemic.

Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from I4 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions (n = 140 [approximately 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.

ELISA and immunoblotting were applied for the characterization of somatic antigens from Absidia corymbifera. Immunoblotting revealed major antigenic bands at 11 to 81 kDa. The ELISA showed some crossreactivity towards somatic antigens from other fungi. However, the crossreactivity was especially observed with somatic antigens from other fungi of the zygomycetes. The ELISA and immunoblotting assays were applied to urine samples from two groups of 3 calves each systemically infected with A. corymbifera and Aspergillus fumigatus, respectively. The immunoreactivity of the urine samples was similar by the two assays. Somatic antigens were demonstrated in the urine of all three calves infected with A.corymbifera, whereas only one of the calves with systemic aspergillosis was antigen positive. The level of antigen in the positive urine samples varied from 50 to 210 ng/ml.

A novel method for diagnosing bovine aspergillosis and zygomycosis is described. Rabbit hyperimmune antisera raised against somatic antigens of Aspergillus fumigatus and Absidia corymbifera were used in crossed immunoelectrophoresis with supernatants from disintegrated tissues from acute necrohaemorrhagic mycotic lesions from cattle. The method specifically identified 4 of 5 lesions with aspergillosis and 2 of 5 lesions with zygomycosis. One lesion dually infected with aspergillosis and zygomycosis was negative. The method worked with unabsorbed sera, was specific, and required only standard electrophoretic equipment. It can therefore supplement chemical detection of fungi in tissues in the diagnosis of bovine aspergillosis and zygomycosis.

Four strains of Mortierella wolfii isolated from cattle in Britain were compared in pathogenicity and toxigenicity with a strain isolated from a cow with the mycotic abortion-pneumonia syndrome in New Zealand. All strains produced acute lethal infection in rabbits after intravenous inoculation of mycelial suspensions and all produced subacute mycotic encephalitis in mice after intracerebral injection. They also produced an acid-stable, heat- and trypsin-labile toxin in vitro. The action of the toxin was exerted mainly on the kidneys in rabbits and mice and produced effects distinct from those resulting from infection with M. wolfii.

Of several media tested for their ability to induce asexual reproduction in confirmed or suspected isolates of Mortierella wolfii obtained from cattle in various geographical locations, only silage extract agar worked consistently and rapidly. Its use should provide a simple reliable culturing procedure to assist with identification of clinical isolates of this fungus. Temperature growth response curves and electrophoretic patterns of soluble protein extracts of these isolates with characteristic morphological features of M. wolfii indicate substantial variation amongst them.

Pilobolus spp. were recovered from all fecal samples collected from an elk (Cervus elaphus nelsoni) herd in Yellowstone National Park (USA) with a high prevalence of Dictyocaulus viviparus infection. Pilobolus spp. have been shown to be important in the epizootiology of D. viviparus infections in cattle because these fungi aid in dissemination of larvae away from feces to areas where animals are more likely to ingest them, and protect larvae against dehydration and thus prolong survival. The same mechanism of dissemination of D. viviparus larvae may play a role in the epizootiology of these infections in elk.