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Journal of Clinical Microbiology Jan 2023There has been significant increase in the use of molecular tools for the diagnosis of invasive aspergillosis (IA) and mucormycosis. However, their range of detection...
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 (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.
Topics: Humans; Mucormycosis; Multiplex Polymerase Chain Reaction; Coinfection; Workflow; COVID-19; Aspergillosis; Mucorales; Invasive Fungal Infections; Aspergillus; Sequence Analysis, DNA; DNA; DNA, Fungal; COVID-19 Testing
PubMed: 36533925
DOI: 10.1128/jcm.01409-22 -
A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.International Journal of Molecular... Dec 2022Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of...
Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., , , , and ) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.
Topics: Humans; Mucorales; Real-Time Polymerase Chain Reaction; Mucormycosis; RNA, Ribosomal, 28S; Immunocompromised Host
PubMed: 36499395
DOI: 10.3390/ijms232315066 -
Frontiers in Microbiology 2022To explore the potential mechanism of improving enzymatic activities in medium-temperature (MTD) by inoculation functional isolates, we inoculated a single strain of ,...
To explore the potential mechanism of improving enzymatic activities in medium-temperature (MTD) by inoculation functional isolates, we inoculated a single strain of , and the microbiota composed of and in MTD to investigate the association between the response of the functional microbiota and the enzymatic activity. The results showed that the bacterial community of MTD might be more sensitive to bioturbation than the fungal community, and the indigenous microbiota responded to the single strain more than to the microbiota. Moreover, the differential microorganisms mainly included , , and between the conventional and fortified samples. Notably, the composition of functional microbiota related to liquefying activity (LA) and saccharifying activity (SA) were significantly different, changing from and to , , and . That might be closely related to the effect of the bioturbation on LA (31.33%) and SA (43.54%) associated microorganisms was more tellingly. Furthermore, the relative abundance changes of bioturbation-sensitive modules in the co-occurrence network might also lead to the difference in enzymatic activities. Therefore, the LA and SA of MTD were improved by bioturbation significantly. These results provide diverse insights into the exogenous functional isolates to regulate the MTD microbiota and improve enzymatic activities.
PubMed: 36458186
DOI: 10.3389/fmicb.2022.1047041 -
Bioengineering (Basel, Switzerland) Oct 2022Morphology plays an important role in the fermentation bioprocess of filamentous fungi. In this study, we investigated the controlling strategies of morphology that...
Morphology plays an important role in the fermentation bioprocess of filamentous fungi. In this study, we investigated the controlling strategies of morphology that improved the efficiency of lipase (RML) production using a high-yield First, the inoculated spore concentrations were optimized in seed culture, and the RML activity increased by 43.4% with the well-controlled mycelium pellets in both ideal sizes and concentrations. Then, the initial nitrogen source and agitation strategies were optimized to regulate the morphology of in a 5 L bioreactor, and the established stable fermentation system increased the RML activity to 232.0 U/mL, combined with an increase in total RML activity from 98,080 U to 487,179 U. Furthermore, the optimized fermentation strategy was verified by a high-yield and achieved an additional improvement of RML activity, up to 320.0 U/mL. Moreover, this optimized fermentation bioprocess was successfully scaled up to a 50 L bioreactor, and the RML activity reached 550.0 U/mL. This work has established a stable precision fermentation bioprocess for RML production by in bioreactors, and the controlling strategy developed in this study could potentially be extended to an industrial scale for RML production with high efficiency.
PubMed: 36354521
DOI: 10.3390/bioengineering9110610 -
Journal of Biotechnology Dec 2022Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation....
Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation. Agricultural side products, wheat straw and corn stover and forestry energy plant willow chips were selected as plant biomass sources. The substrates were confronted by pure cultures of Penicillium aurantiogriseum (new isolate from rumen), Trichoderma reesei (DSM768), Gilbertella persicaria (SZMC11086) and Rhizomucor miehei (SZMC11005). In addition to total cellulolytic filter paper degradation activity, the production of endoglucanase, cellobiohydrolase, β-glucosidase enzymes were followed during the pretreatment period, which lasted for 10 days at 37 °C. The products of pretreatments were subsequently tested for mesophilic biogas production in batch reactors. All 4 strains effectively pretreated the lignocellulosic substrates albeit in varying degrees, which was related to the level of the tested hydrolytic enzyme activities. Penicillium aurantiogriseum showed outstanding hydrolytic enzyme production and highest biogas yield from the partially degraded substrates. Corn stover was the best substrate for biomass decomposition and biogas production. Scanning electron microscopy confirmed the deep penetration of fungal hyphae into the lignocellulosic substrate in all cases.
Topics: Biofuels
PubMed: 36273669
DOI: 10.1016/j.jbiotec.2022.10.013 -
Polymers Sep 2022During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the...
During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the simplicity of the biodegradation of natural polymers. The protein retention by and release from the polysaccharide gel network is regulated by geometry and physical interactions of protein with the matrix. In the present work, we studied the molecular details of interactions between κ-carrageenan and three lipases, namely the lipases from , , and -which differ in their size and net charge-upon protein immobilization in microparticles of polysaccharide gel. The kinetics of protein release revealed the different capability of κ-carrageenan to retain lipases, which are generally negatively charged; that was shown to be in line with the energy of interactions between polysaccharides and positively charged epitopes on the protein surface. These data create a platform for the novel design of nanocarriers for biomedical probes of enzymatic origin.
PubMed: 36236018
DOI: 10.3390/polym14194071 -
Cureus Aug 2022An elderly female presented to the emergency department with a right-sided facial droop and headache for two weeks. Investigations revealed poorly controlled diabetes,...
An elderly female presented to the emergency department with a right-sided facial droop and headache for two weeks. Investigations revealed poorly controlled diabetes, and the patient was found to be in diabetic ketoacidosis. Maxillofacial computed tomography (CT) demonstrated right postseptal cellulitis with concern for acute invasive fungal sinusitis. The patient was taken to the operating room for orbital surgical exploration and antrostomy. Surgical pathology revealed broad hyphae consistent with species, and the patient was diagnosed with mucormycosis. Because the patient was not clinically improving, further imaging was obtained, which showed a large right retroantral phlegmon extending into the cranial fossa and right cavernous sinus, and the patient subsequently underwent surgical debridement. The following postoperative day, the patient was stroke-alerted due to altered mental status and inability to follow commands. She was found to have a small embolic infarct. Due to the poor prognosis of the patient, she was discharged with hospice. Mucormycosis is more commonly found in immunocompromised patients, such as those with uncontrolled diabetes mellitus but very rarely does it involve the cranium. This disease process is very important to recognize early due to high morbidity and mortality rates and devastating outcomes.
PubMed: 36158403
DOI: 10.7759/cureus.28104 -
Journal of Fungi (Basel, Switzerland) Jul 2022Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore,...
Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore, compared the commercial MucorGenius assay (PathoNostics, Maastricht) targeting five genera of Mucorales to our in-house qPCR targeting spp., spp. and spp. To assess their analytical sensitivity, 25 frozen leftover serum specimens, which had already tested positive based on our in-house assay, were selected. These sera were from 15 patients with probable or proven mucormycosis. For analytical specificity, 0.5 pg from 15 purified fungal DNAs from nine different Mucorales genera were spiked into pooled qPCR-negative leftover serum specimens. All samples were tested in parallel with both assays and the quantitative cycles (Cq) were compared. A total of 13/25 (52%) serum samples were amplified by one of the two assays with only four of them detected with the MucorGenius assay. In spiked specimens, all targeted strains were successfully amplified by our in-house qPCR. The MucorGenius assay was not able to detect but successfully amplified all other species targeted by the kit and two additional non-targeted species ( and ). The MucorGenius assay showed lower analytical sensitivity compared to our in-house assay. Indeed, the MucorGenius assay amplified more species, as expected, but showed a decreased detection of the frequent species .
PubMed: 36012775
DOI: 10.3390/jof8080786 -
EFSA Journal. European Food Safety... Aug 2022The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain DSM 29547 by Chr. Hansen. The food enzyme is free from viable cells of...
The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain DSM 29547 by Chr. Hansen. The food enzyme is free from viable cells of the production organism. It is intended to be used in dairy processing for cheese production. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.26 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 618 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,400. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.
PubMed: 35978613
DOI: 10.2903/j.efsa.2022.7457 -
Infection and Drug Resistance 2022Long-term chemotherapy and immunosuppressants in acute myeloid leukaemia (AML) patients can result in a high risk of opportunistic infections. is an opportunistic...
Long-term chemotherapy and immunosuppressants in acute myeloid leukaemia (AML) patients can result in a high risk of opportunistic infections. is an opportunistic pathogen that exists in nature, but infection caused by is rare in the clinic. Notably, the sensitivity and detection time of conventional diagnostic tools for this fungus usually falls short of the needs of clinical diagnosis, resulting in treatment failure. Currently, metagenomics next-generation sequencing (mNGS) has played an important role in the detection of pathogens. Here, we report a case of pneumonia in a haematopoietic stem cell transplantation (HSCT) patient, detected by the mNGS method.
PubMed: 35965849
DOI: 10.2147/IDR.S376045