Water Extracts from Industrial Hemp Waste Inhibit the Adhesion and Development of Candida Biofilm and Showed Antioxidant Activity on HT-29 Colon Cancer Cells.

The evolution of regulatory perspectives regarding the health and nutritional properties of industrial hemp-based products (Cannabis sativa L.) has pushed research to focus on the development of new methods for both the extraction and formulation of the bioactive compounds present in hemp extracts. While the psychoactive and medicinal properties of hemp-derived cannabinoid extracts are well known, much less has been investigated on the functional and antimicrobial properties of hemp extracts. Within the hemp value chain, various agricultural wastes and by-products are generated. These materials can be valorised through eco-innovations, ultimately promoting sustainable economic development. In this study, we explored the use of waste from industrial light cannabis production for the extraction of bioactive compounds without the addition of chemicals. The five extracts obtained were tested for their antimicrobial activity on both planktonic and sessile cells of pathogenic strains of the Candida albicans, Candida parapsilosis, and Candida tropicalis species and for their antioxidant activity on HT-29 colon cancer cells under oxidative stress. Our results demonstrated that these extracts display interesting properties both as antioxidants and in hindering the development of fungal biofilm, paving the way for further investigations into the sustainable valorisation of hemp waste for different biomedical applications.

Metabolic Syndrome and Its Components Have a Different Presentation and Impact as Cardiovascular Risk Factors in Psoriatic and Rheumatoid Arthritis.

Patients with chronic inflammatory arthritis have a higher cardiovascular (CV) risk than the general population. Traditional CV risk factors are clearly implicated, while the impact of metabolic syndrome (MetS) is less defined. The aim of this study was to compare MetS prevalence and impact on the CV risk in psoriatic arthritis (PsA) versus rheumatoid arthritis (RA). A retrospective analysis of real-world data of PsA and RA patients referred to a rheumatology clinic was conducted. The following data were extracted and compared: demographic data; clinical data; presence of traditional CV risk factors and MetS. Univariate and multivariate models were used to compare the impact of MetS and its components in patients with PsA versus RA. Overall, 170 patients were included (PsA: 78; RA; 92). The two groups differed significantly in mean age, disease duration, and presence of MetS, while other variables were comparable. Univariate and multivariate analysis identified distinct predictors of MetS in PsA (hypertension) and RA (dyslipidemia). The history of CV events was similar in the two groups. Predictors of CV events were MetS and most of its components in PsA, while dyslipidemia was the strongest predictor in RA. These associations were stronger in PsA than in RA. In conclusion, the impact of MetS and its components is different in PsA and RA. The association of these risk factors with CV events is stronger in PsA than in RA. This suggests the implication of different mechanisms, which may require distinct strategies for the prevention of CV events in PsA and RA.

Evolution of Rheumatoid-Arthritis-Associated Interstitial Lung Disease in Patients Treated with JAK Inhibitors: A Retrospective Exploratory Study.

BACKGROUND: The aim of this multicenter retrospective study was to investigate the effectiveness and safety of the available JAK-inhibitors (JAKi) in patients with rheumatoid arthritis (RA) and interstitial lung disease (ILD). METHODS: We retrospectively analyzed patients with classified RA and RA-ILD undergoing JAKi in 6 Italian tertiary centers from April 2018 to June 2022. We included patients with at least 6 months of active therapy and one high-resolution chest tomography (HRCT) carried out within 3 months of the start of JAKi treatment. The HRCT was then compared to the most recent one carried out within 3 months before the last available follow-up appointment. We also kept track of the pulmonary function tests. RESULTS: We included 43 patients with RA-ILD and 23 males (53.48%) with a median age (interquartile range, IQR) of 68.87 (61.46-75.78) treated with JAKi. The median follow-up was 19.1 months (11.03-34.43). The forced vital capacity remained stable in 22/28 (78.57%) patients, improved in 3/28 (10.71%) and worsened in 3/28 (10.71%). The diffusing capacity of lung for carbon monoxide showed a similar trend, remaining stable in 18/25 (72%) patients, improving in 2/25 (8%) and worsening in 5/25 (20%). The HRCT remained stable in 37/43 (86.05) cases, worsened in 4/43 (9.30%) and improved in the last 2 (4.65%). DISCUSSION: This study suggests that JAKi therapy might be a safe therapeutic option for patients with RA-ILD in a short-term follow-up.

Renewing Lost Genetic Variability with a Classical Yeast Genetics Approach.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis.

Microbial communities play key roles both for humans and the environment. They are involved in ecosystem functions, maintaining their stability, and provide important services, such as carbon cycle and nitrogen cycle. Acting both as symbionts and as pathogens, description of the structure and composition of these communities is important. Metabarcoding uses ribosomal DNA (rDNA) (eukaryotic) or rRNA gene (prokaryotic) sequences for identification of species present in a site and measuring their abundance. This procedure requires several technical steps that could be source of bias producing a distorted view of the real community composition. In this work, we took advantage of an innovative "long-read" next-generation sequencing (NGS) technology (MinION) amplifying the DNA spanning from the internal transcribed spacer (ITS) to large subunit (LSU) that can be read simultaneously in this platform, providing more information than "short-read" systems. The experimental system consisted of six fungal mock communities composed of species present at various relative amounts to mimic natural situations characterized by predominant and low-frequency species. The influence of the sequencing platform (MinION and Illumina MiSeq) and the effect of different reference databases and marker sequences on metagenomic identification of species were evaluated. The results showed that the ITS-based database provided more accurate species identification than LSU. Furthermore, a procedure based on a preliminary identification with standard reference databases followed by the production of custom databases, including only the best outputs of the first step, is proposed. This additional step improved the estimate of species proportion of the mock communities and reduced the number of ghost species not really present in the simulated communities. IMPORTANCE Metagenomic analyses are fundamental in many research areas; therefore, improvement of methods and protocols for the description of microbial communities becomes more and more necessary. Long-read sequencing could be used for reducing biases due to the multicopy nature of rDNA sequences and short-read limitations. However, these novel technologies need to be assessed and standardized with controlled experiments, such as mock communities. The interest behind this work was to evaluate how long reads performed identification and quantification of species mixed in precise proportions and how the choice of database affects such analyses. Development of a pipeline that mitigates the effect of the barcoding sequences and the impact of the reference database on metagenomic analyses can help microbiome studies go one step further.

Metabolomic and Proteomic Changes in Candida albicans Biofilm in Response to Zosteric Acid Treatment.

Zosteric acid (ZA) is a secondary metabolite of the seagrass Zostera marina, with antibiofilm activity against fungi. Information concerning its mechanisms of action is lacking and this limits the development of more potent derivatives based on the same target and activity structure. The aim of this work was to investigate the ZA mode of action by analyzing the metabolic status of Candida albicans biofilm and its protein expression profile upon ZA treatment. Fourier-Transform Infrared Spectroscopy confirmed that ZA modified the metabolomic response of treated cells, showing changes in the spectral regions, mainly related to the protein compartment. Nano Liquid Chromatography-High-Resolution Mass Spectrometry highlighted that 10 proteins were differentially expressed in the C. albicans proteome upon ZA treatment. Proteins involved in the biogenesis, structure and integrity of cell walls as well as adhesion and stable attachment of hyphae were found downregulated, whereas some proteins involved in the stress response were found overexpressed. Additionally, ZA was involved in the modulation of non-DNA-based epigenetic regulatory mechanisms triggered by reactive oxygen species. These results partially clarified the ZA mechanism of action against fungi and provided insight into the major C. albicans pathways responsible for biofilm formation.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

One year in review 2021: fibromyalgia.

Fibromyalgia (FM) is a syndrome of unknown aetiology characterised by chronic pain, fatigue, and disturbed sleep. This review presents and summarises the 2020 literature on FM by retrieving all articles indexed in PubMed between 1 January 2020 and 31 December 2020. The attention of the scientific community towards FM is constantly growing, and this year's review is focused on the diagnostic, pathogenetic and therapeutic aspects of this syndrome. In particular, the treatment options for FM, both pharmacological and non-pharmacological, have been extensively studied.

How does temperature trigger biofilm adhesion and growth in Candida albicans and two non-Candida albicans Candida species?

BACKGROUND: Biofilm formation on biotic and abiotic surfaces is finely regulated by genetic factors but also by oxygen concentration, pH, temperature and other environmental factors, already extensively explored for bacterial biofilms. Much less is known about fungal biofilm, that is considered a virulence factor for Candida pathogenic species among the few fungal species able to grow and survive at high temperatures such as 37°C as well as those induced by fever. The resistance to high temperatures coupled with the ability to form biofilm are threatening factors of these fungal species that could severely impact at an epidemiological level. OBJECTIVES: In this framework, we decided to study the thermal tolerance of biofilms formed by three medical relevant species such as Candida albicans and two non-Candida albicans Candida species. METHODS: Thirty nosocomial strains were investigated for their ability to adhere and grow in proximity and over body temperature (from 31 to 43°C), mimicking different environmental conditions or severe febrile-like reactions. RESULTS: Candida sessile cells reacted to different temperatures showing a strain-specific response. It was observed that the attachment and growth respond differently to the temperature and that mechanism of adhesion has different outputs at high temperature than the growth. CONCLUSIONS: This strain-dependent response is probably instrumental to guarantee the best success to cells for the infection, attachment and growth to occur. These observations reinforce the concept of temperature as a major trigger in the evolution of these species especially in this period of increasing environmental temperatures and excessive domestic heating.

The Rheumatology Drugs for COVID-19 Management: Which and When?

INTRODUCTION: While waiting for the development of specific antiviral therapies and vaccines to effectively neutralize the SARS-CoV2, a relevant therapeutic strategy is to counteract the hyperinflammatory status, characterized by an increase mainly of interleukin (IL)-1ß, IL-2, IL-6, IL-7, IL-8, and tumor necrosis factor (TNF)-α, which hallmarks the most severe clinical cases. 'Repurposing' immunomodulatory drugs and applying clinical management approved for rheumatic diseases represents a game-changer option. In this article, we will review the drugs that have indication in patients with COVID-19, including corticosteroids, antimalarials, anti-TNF, anti-IL-1, anti-IL-6, baricitinib, intravenous immunoglobulins, and colchicine. The PubMed, Medline, and Cochrane Library databases were searched for English-language papers concerning COVID-19 treatment published between January 2020 and October 2020. Results were summarized as a narrative review due to large heterogeneity among studies. In the absence of specific treatments, the use of immunomodulatory drugs could be advisable in severe COVID-19 patients, but clinical outcomes are still suboptimal. An early detection and treatment of the complications combined with a multidisciplinary approach could allow a better recovery of these patients.

What Is the Best Lens? Comparing the Resolution Power of Genome-Derived Markers and Standard Barcodes.

Fungal species delimitation was traditionally carried out with multicopy ribosomal RNA (rRNA) genes, principally for their ease of amplification. Since the efficacy of these markers has been questioned, single-copy protein-encoding genes have been proposed alone or in combination for Multi-Locus Sequence Typing (MLST). In this context, the role of the many sequences obtained with Next-Generation Sequencing (NGS) techniques, in both genomics and metagenomics, further pushes toward an analysis of the efficacy of NGS-derived markers and of the metrics to evaluate the marker efficacy in discriminating fungal species. This paper aims at proposing MeTRe (Mean Taxonomic Resolution), a novel index that could be used both for measuring marker efficacy and for assessing the actual resolution (i.e., the level of separation) between species obtained with different markers or their combinations. In this paper, we described and then employed this index to compare the efficacy of two rRNAs and four single-copy markers obtained from public databases as both an amplicon-based approach and genome-derived sequences. Two different groups of species were used, one with a pathogenic species of Candida that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the sensu stricto group of the genus Saccharomyces, included close species and interspecific hybrids. The results showed the ability of MeTRe to evaluate marker efficacy in general and genome-derived markers specifically.

Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus Candida.

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.

Homoplasy as an Auxiliary Criterion for Species Delimitation.

Homoplasy is a sort of noise in phylogenetic reconstructions, due to the accumulation of backmutations, convergent evolution and horizontal gene transfer (HGT), which is considered the major trigger of homoplasy in microorganism for its massive presence. It is also known that homoplasy increases with the complexity of the tree with both real and simulated data. In this paper, we analyzed the variation of homoplasy with the two widely used taxonomic markers ITS and LSU in four taxonomic models characterized by differences in the intra-specific distances. An algorithm (HomoDist) was developed to analyze the homoplasy index (HI) variation upon addition of a single element (strain or species) in increasing distance from a starting element. This algorithm allows to follow changes of the consistency index (CI), complementary to the HI, with the increase of the number of taxa and with the increase of the distance among elements. Results show that homoplasy increases-as expected-with the number of taxa, but also as a function of the overall distance among species, often with an almost linear relationship between distance and HI. No HI change was observed in trees with few taxa spanning through short distances, indicating that this noise is not prohibitive in this context, although the analysis of the ratio between HI and distance can be recommended as a criterion for tree acceptance. The absence of large changes of the HI within the species, and its increase when new species are added by HomoDist, suggest that homoplasy variation can be used as an auxiliary test in distance-based species delimitation with any type of marker.

Efficacy of cyclophosphamide in treating connective tissue disease-related interstitial lung diseases.

Interstitial lung diseases (ILDs) are some of the first and most serious complications of connective tissue diseases (CTDs). However, the pathogenesis of CTD-related ILDs (CTD-ILDs) is still unclear and their treatment often depends on functional and radiographic disease progression as well as on patient age and comorbidities. It can be difficult to manage CTD-ILDs due to their heterogeneous nature, the lack of robust therapeutic data, and the few well-defined outcome measures. This review focuses on cyclophosphamide due to its crucial role in the treatment of systemic sclerosis-related ILD, particularly in the case of patients with progressive ILD. This narrative review was performed using PubMed, Medline, and Cochrane Library databases to retrieve English language papers published between 2000 and April 2020 concerning the treatment of CTD-ILDs with cyclophosphamide.

The effect of drugs used in rheumatology for treating SARS-CoV2 infection.

INTRODUCTION: SARS-CoV-2 is a novel coronavirus that was first isolated from a group of patients hospitalized with pneumonia in China at the end of 2019, and, in February 2020, the syndrome it caused was named coronavirus disease 2019 (COVID-19) by the World Health Organization. In the absence of specific antiviral treatments capable of neutralizing the etiological agent, one therapeutic approach is to control the cytokine storm responsible for the most severe forms of the disease. The characteristic cytokine profile of severely affected patients is increased levels of interleukin (IL)-1ß, IL-2, IL-6, IL-7, IL-8, and tumor necrosis factor alpha (TNF-α). AREAS COVERED: This article discusses the pathogenesis of COVID-19 as a rationale for using the biological and targeted synthetic drugs used in rheumatology (anti-TNF, anti-IL-1 and anti-IL-6 agents and baricitinib) to treat the disease, and provides key information concerning their potential benefits and adverse effects. EXPERT OPINION: Interleukin inhibition seems to be a promising means of treating COVID-19 patients when respiratory function declines (or even earlier) if there are laboratory data indicating the presence of a cytokine storm because the interleukins are key drivers of inflammation. However, it is important to consider the risks and benefits of biological agents carefully, and critically analyze the evidence concerning their use in COVID-19 patients.

Do Metabolomics and Taxonomic Barcode Markers Tell the Same Story about the Evolution of Saccharomyces sensu stricto Complex in Fermentative Environments?

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the importance of physiology and paving the way to the DNA-based taxonomy. The hypothesis of reconnecting taxonomy with specific traits from phylogenies has been successfully explored for Bacteria and Archaea, suggesting that a similar route can be traveled for yeasts. In this framework, thirteen single copy loci were used to investigate the predictability of complex Fourier Transform InfaRed spectroscopy (FTIR) and High-performance Liquid Chromatography-Mass Spectrometry (LC-MS) profiles of the four historical species of the Saccharomyces sensu stricto group, both on resting cells and under short-term ethanol stress. Our data show a significant connection between the taxonomy and physiology of these strains. Eight markers out of the thirteen tested displayed high correlation values with LC-MS profiles of cells in resting condition, confirming the low efficacy of FTIR in the identification of strains of closely related species. Conversely, most genetic markers displayed increasing trends of correlation with FTIR profiles as the ethanol concentration increased, according to their role in the cellular response to different type of stress.

Toxicological considerations in the treatment of axial spondylo-arthritis.

INTRODUCTION: The first-line treatment of axial spondyloarthritis (SpA) is with non-steroidal anti-inflammatory drugs (NSAIDs) and is followed by tumor necrosis factor (TNF) inhibitors (the main treatment for patients not responding to NSAIDs) or drugs targetting the IL-23/IL-17 pathway. The efficacy of disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate and sulfasalazine (SSZ) has not been demonstrated, although SSZ can be considered in patients with concomitant peripheral arthritis. AREAS COVERED: This review describes the beneficial and toxicological effects of the drugs used to treat axial SpA. EXPERT COMMENTARY: Growing concerns about the safety of anti-TNF drugs underline the need to ensure that all clinicians are capable of taking appropriate preventive action and adequately treating affected patients.

Potential Role of Anti-interleukin (IL)-6 Drugs in the Treatment of COVID-19: Rationale, Clinical Evidence and Risks.

The epidemic due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been spreading globally, raising increasing concerns. This public health emergency has triggered a race to find medications to improve the prognosis of this disease. There is currently great interest in drug repositioning to manage SARS-CoV-2 infection, that is, the evaluation of the potential benefits of a drug that has already been proven safe and effective in humans for other approved indications. As interleukin-6 (IL-6) acts as a key driver of the inflammation associated with coronavirus disease 2019 (COVID-19), IL-6 and IL-6 receptor (IL-6R) inhibition appear to be promising targets for the treatment of COVID-19 patients. It is important to critically analyze the available evidence concerning the use of the available anti-IL-6 (siltuximab) and anti-IL-6R (tocilizumab and sarilumab) agents in COVID-19 patients, in terms of both benefit and risk. In this review, the pathogenesis of the cytokine storm induced by COVID-19, the role of IL-6 in this cytokine storm, the rationale for the use of anti-IL-6 agents, and key information on potential benefits and safety monitoring of these biologicals in COVID-19 patients is discussed.

Delta-Integration of Single Gene Shapes the Whole Metabolomic Short-Term Response to Ethanol of Recombinant Saccharomyces cerevisiae Strains.

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial Saccharomyces cerevisiae 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains' tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.

Candida palmioleophila isolation in Italy from two cases of systemic infection, after a CHROMagar and Vitek system mis-identification as C. albicans.

A correct, fast, reliable identification method is pivotal in nosocomial environments to guide treatment strategies, whereas misidentification might lead to treatment failure. For routine identifications the Vitek system and CHROMagar are widely used but not always reliable, especially now with an increasing number of new emerging fungal pathogens that need careful identification. Here we describe two cases of candidemia, due to Candida palmioleophila previously misidentified as Candida albicans by using the Vitek2 system and CHROMagar. The first case is a 54-year-old man with an infected ulcer in the lower right limb, treated with a targeted therapy using a central venous catheter (CVC). After two months he developed a CVC-related candidemia MDR identified as C. albicans. The second case is a 2-month-old male baby that was admitted to the neonatal unit with acute respiratory failure due to a severe community-acquired bilateral pneumonia; blood cultures were all positive for C. albicans MDR. The isolated strains where re-identified with Maldi-Tof and DNA sequencing as C. palmioleophila. From the identification point of view, CHROMagar can be clearly misleading, especially because CHROMagar types currently available are not designed to discriminate new emerging species, suggesting that systems other than MALDI-TOF and marker sequencing may be inadequate even for routine identification and could contribute to producing misleading identifications and therapeutically wrong practices, leading to failures and patient death.