Background:Mucormycosis is a highly aggressive and destructive angio-invasive infection caused by fungi of the order Mucorales. Diagnosis currently relies on lengthy and insensitive culture of biopsy specimens, as well as time-consuming histopathological examination of tissue samples, which lacks specificity. Case presentation:This case study describes an unusual presentation of cutaneous mucormycosis in an immunocompetent 49-year-old man following a traumatic farming injury to the hand. Despite appearing clinically well and having wounds that looked macroscopically clean after repeated debridement, the patient had a persistent infection caused by the Mucorales fungus, Lichtheimia ramosa. Diagnosis was delayed due to low clinical suspicion and routine homogenisation of biopsy samples, which reduced the sensitivity of conventional fungal culture and microscopy. Early surgical debridement combined with antifungal therapy was essential; however, treatment was complicated by amphotericin B-associated nephrotoxicity, necessitating a switch to posaconazole. The patient ultimately achieved wound healing with preserved hand function. Conclusion:The case demonstrates key challenges in diagnosing cutaneous mucormycosis, particularly in immunocompetent patients with environmentally contaminated wounds. The study highlights the need for improved awareness, optimal tissue handling, and more specific diagnostics for mucormycosis. We demonstrate the successful clinical use of a Mucorales-specific monoclonal antibody (mAb TG11), both as an immunohistochemistry stain and within a rapid lateral-flow device (LFD), to detect signature molecules of Mucorales infection in homogenised tissue samples, thereby improving the accuracy and speed of detection.

Fungal morphogenesis is a major driver of disease outcome. For the opportunistic fungal pathogen Cryptococcus neoformans, extreme morphological heterogeneity within the lung environment drives dissemination, immune evasion, and drug resistance. One such morphotype is the small, oval, titanide (2-3 μm). This poorly understood cell type is prevalent in lung histology and under in vitro conditions that mimic the host environment to generate cellular heterogeneity. Titanides appear after 24 hours post-induction and by 72 hours are the dominant morphotype. Despite their prevalence and the significance of cryptococcal morphogenesis for virulence, their origin remains unclear and their biology unstudied. Using TEM and fluorescence microscopy we demonstrate that titanides display distinct morphological features; they possess thin cell walls and capsules with reduced Pathogen Associated Molecular Pattern exposure and altered distribution. These features distinguish titanides from previously described C. neoformans ′small cells′ such as in vivo seed cells (4-6 μm, high cell-wall mannan content) and micro cells (round, <1 μm, with thick cell walls). Using microfluidics, we answer key questions pertaining to their origin and cell fate and further our understanding of C. neoformans typical cell and titanide morphological plasticity in host-relevant conditions. Interestingly, despite their thin cell walls, we show that titanides display an increased resistance to cell wall/membrane stressors and are an effective infectious propagule in a murine model. Together, these findings establish a definition and essential characterisation of the titanide morphotype and provide new insight into a key player in C. neoformans pathogenesis. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 211241/Z/18/Z, 337892/Z/25/Z Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/W002760/1 Medical Research Council, https://ror.org/03x94j517, MR/Z504798/1, MR/N006364/2, MR/V033417/1 NIHR Exeter Biomedical Research Centre, 5411520

Aspergillosis is a disease caused by the filamentous fungus Aspergillus spp. with a spectrum of clinical presentation that includes invasive and noninvasive forms. The invasive clinical presentation of aspergillosis most frequently affects people with compromised immune systems. In patients with oncohematologic pathology, invasive lung aspergillosis is a significant opportunistic mycosis, because it occurs frequently and has a major impact on morbidity, mortality, and high costs. The global problem of antimicrobial resistance, to which improper use of antifungals contributes, has put Aspergilus spp. in the spotlight, so it is important to generate guidelines for guidance in the proper use of antifungals in the management of invasive lung aspergillosis, to obtain better clinical outcomes and promote rational use of antifungals. This guideline contains recommendations for diagnosing and treating invasive lung aspergillosis in patients with oncohematologic disease, based on evidence and defined through a participatory process of expert consensus, for the Latin American context.

Candidemia is the predominant form of invasive candidiasis and the most frequently occurring serious fungal infection in critically ill patients in Intensive Care Units (ICU). Studies carried out in Latin America reveal a higher incidence of candidemia and higher mortality rates when compared to North America or Europe. This highlights the need to develop guidelines for correctly diagnosing and treating candidemia in critically ill patients in the ICU. These guidelines are part of the efforts to implement antifungal optimization programs in the region to obtain better clinical outcomes and promote rational antifungal use. This evidence-based clinical standard, established through expert consensus for the Latin American context, contains recommendations and algorithms for diagnosing and treating candidemia in critically ill ICU patients.

BACKGROUND:Mucormycosis is an aggressive invasive fungal infection caused by molds in the order Mucorales. Early diagnosis is key to improving patient prognosis, yet it relies on insensitive culture or nonspecific histopathology. A pan-Mucorales-specific monoclonal antibody (mAb), TG11, was recently developed. Here, we investigate the spatiotemporal localization of the antigen and specificity of the mAb for immunohistochemistry. METHODS:We used immunofluorescence microscopy to assess antigen localization in 11 Mucorales species of clinical importance and live imaging of Rhizopus arrhizus germination. Immunogold transmission electron microscopy revealed the subcellular location of mAb TG11 binding. Finally, we performed immunohistochemistry of R arrhizus in an ex vivo murine lung infection model alongside lung infection by Aspergillus fumigatus. RESULTS:Immunofluorescence revealed TG11 antigen production at the emerging hyphal tip and along the length of growing hyphae in all Mucorales except Saksenaea. Time-lapse imaging revealed early antigen exposure during spore germination and along the growing hypha. Immunogold transmission electron microscopy confirmed mAb TG11 binding to the hyphal cell wall only. The TG11 mAb stained Mucorales but not Aspergillus hyphae in infected murine lung tissue. CONCLUSIONS:TG11 detects early hyphal growth and has valuable potential for diagnosing mucormycosis by enhancing discriminatory detection of Mucorales in tissue.

The burden of opportunistic infections (OIs) remains high among people living with HIV (PLWH) in Mexico, despite improvements in mortality worldwide. Reporting the current access to diagnostics of OIs in Mexican Health Care Centers offering health-care services to PLWH. An online questionnaire was sent to public health care facilities providing HIV care in Mexico. We evaluated capacities to 1) identify individuals with advanced-HIV, and 2) local and/or on-site access to: point-of-care assays, imaging studies, histological analysis, and microbiology tests useful to diagnose a wide variety of OIs. In 2022, 46 centers answered the questionnaire, from 23/32 (71.8

Introduction: Escalation of chemical disinfection during the COVID-19 pandemic has raised occupational hazard concerns. Alternative and potentially safer methods such as ultraviolet-C (UVC) irradiation and ozone have been proposed, notwithstanding the lack of standardized criteria for their use in the healthcare environment. Aim :Compare the virucidal activity of 70% ethanol, sodium dichloroisocyanurate (NaDCC), chlorhexidine, ozonated water, UVC-222 nm, UVC-254 nm against three SARS-CoV-2 variants of concern cultured in vitro. Methods: Inactivation of three SARS-CoV-2 variants (alpha, beta, gamma) by the following chemical methods was tested: ethanol 70%, NaDCC (100 ppm, 500 ppm, 1000 ppm), chlorhexidine (2%, 1% and 0.5%), ozonated water 7 ppm. For irradiation, a je2Care 222nm UVC Lamp was compared to a Sylvania G15 UV254 nm lamp. Results: Viral inactivation by >3 log was achieved with ethanol, NaDCC and chlorhexidine. The minor virucidal effect of ozonated water was <1 log. Virus treatment with UVC-254 nm reduced viral activity by 1-5 logs with higher inactivation after exposure for 3 minutes compared to 6 seconds. For all three variants, under equivalent conditions, exposure to UVC-222 nm did not achieve time-dependent inactivation as was observed with treatment with UVC-254 nm. Conclusion: The virucidal activity on replication-competent SARS-CoV-2 by conventional chemical methods, including chlorhexidine at concentrations as low as 0.5%, was not matched by UVC irradiation, and to an even lesser extent by ozonated water treatment.

Background: Damage due to respiratory viruses increases the risk of bacterial and fungal coinfections and superinfections. High rates of invasive aspergillosis are seen in severe influenza and COVID-19. This report describes CAPA cases diagnosed during the first wave in the biggest reference centre for severe COVID-19 in Mexico.Objectives: To describe the clinical, microbiological and radiological characteristics of patients with invasive pulmonary aspergillosis associated with critical COVID-19, as well as to describe the variables associated with mortality.Methods: This retrospective study identified CAPA cases among individuals with COVID-19 and ARDS, hospitalised from 1 March 2020 to 31 March 2021. CAPA was defined according to ECMM/ISHAM consensus criteria. Prevalence was estimated. Clinical and microbiological characteristics including bacterial superinfections, antifungal susceptibility testing and outcomes were documented.Results: Possible CAPA was diagnosed in 86 patients among 2080 individuals with severe COVID-19, representing 4.13% prevalence. All CAPA cases had a positive respiratory culture for Aspergillus species. Aspergillus fumigatus was the most frequent isolate (64%, n = 55/86). Seven isolates (9%, n = 7/80) were resistant to amphotericin B (A. fumigatus n = 5/55, 9%; A. niger, n = 2/7, 28%), two A. fumigatus isolates were resistant to itraconazole (3.6%, n = 2/55). Tracheal galactomannan values ranged between 1.2 and 4.05, while serum galactomannan was positive only in 11% (n = 3/26). Bacterial coinfection were documented in 46% (n = 40/86). Gram negatives were the most frequent cause (77%, n = 31/40 isolates), from which 13% (n = 4/31) were reported as multidrug-resistant bacteria. Mortality rate was 60% and worse prognosis was seen in older persons, high tracheal galactomannan index and high HbA1c level.Conclusions: One in 10 individuals with CAPA carry a resistant Aspergillus isolate and/or will be affected by a MDR bacteria. High mortality rates are seen in this population.

ABSTRACT Members of the Meyerozyma guilliermondii species complex are able to cause superficial and life-threatening systemic infections with low susceptibility to azoles and echinocandins. We tested 130 bloodstream M. guilliermondii complex isolates collected from eight Latin American medical centers over 18 years (period 1 = 2000–2008 and period 2 = 2009–2018) to investigate trends in species distribution and antifungal resistance. The isolates were identified by rDNA ITS region sequencing, and antifungal susceptibility tests were performed against fluconazole, voriconazole, anidulafungin, and amphotericin B using the CLSI microbroth method. M. guilliermondii sensu stricto (s.s.; n = 116) was the most prevalent species, followed by Meyerozyma caribbica ( n = 12) and Meyerozyma carpophila ( n = 2). Based on rDNA ITS identification, three clades within M. guilliermondii sensu stricto were characterized (clade 1 n = 94; clade 2 n = 19; and clade 3 n = 3). In the second period of study, we found a substantial increment in the isolation of M. caribbica (3.4% versus 13.8%; P = 0.06) and clade 2 M. guilliermondii s.s. exhibiting lower susceptibility to one or more triazoles. IMPORTANCE Yeast-invasive infections play a relevant role in human health, and there is a concern with the emergence of non- Candida pathogens causing disease worldwide. There is a lack of studies addressing the prevalence and antifungal susceptibility of different species within the M. guilliermondii complex that cause invasive infections. We evaluated 130 episodes of M. guilliermondii species complex candidemia documented in eight medical centers over 18 years. We detected the emergence of less common species within the Meyerozyma complex causing candidemia and described a new clade of M. guilliermondii with limited susceptibility to triazoles. These results support the relevance of continued global surveillance efforts to early detect, characterize, and report emergent fungal pathogens exhibiting limited susceptibility to antifungals.

Malassezia yeast species are the dominant commensal fungal species of the human skin microbiota, but are also associated with inflammatory skin diseases, such as seborrheic dermatitis and atopic eczema (AE). Mala s 1, a β-propeller protein, is an allergen identified in Malassezia sympodialis inducing both IgE and T-cell reactivity in the majority of patients with AE. In this study, we aimed to elucidate the role of Mala s 1 allergen in skin disease. An anti-Mala s 1 antibody was used to investigate the cellular localisation of Mala s 1, the potential of Mala s 1 as a therapeutic target and examine cross-reactivity of the anti-Mala s 1 antibody with human skin. We demonstrate by high pressure freezing electron microscopy and immune-staining that Mala s 1 is located in the cell wall of M. sympodialis yeast cells. Despite the ability of the anti-Mala s 1 antibody to bind to yeast cells, it did not inhibit M. sympodialis growth suggesting Mala s 1 may not be an attractive antifungal target. The Mala s 1 predicted protein sequence was analysed in silico and was found to contain a motif indicative of a KELCH protein, a group of β-propeller proteins. Humans express a large number of KELCH proteins including some that are localised in the skin. To test the hypothesis that antibodies against Mala s 1 cross react with human skin proteins we examined the binding of anti-Mala s 1 antibody to human skin explant samples. Reactivity with the antibody was visualised in the epidermal layer of skin. To further characterise putative human targets recognised by the anti-Mala s 1 antibody, proteins were extracted from immunoblot gel bands and proteomic analysis performed. Several candidate human proteins were identified. To conclude, we propose that Mala s 1 is a KELCH-like β-propeller protein with similarity to human skin proteins and Mala s 1 recognition may trigger the production of cross-reactive responses that contribute to skin diseases associated with M. sympodialis .

Rhizopus arrhizus is a member of a complex of soil-associated fungal species distributed worldwide that cause spoilage of vegetables and fruit. R. arrhizus is also widely used in food industry in the production of fermented food and in other biotechnological industries, such as research and development of new therapeutic compounds. This species has been isolated from plants and animals and can also cause disease in humans.

Candida auris can persist for long periods on hospital surfaces and on the skin. C. auris has the ability to form drug-resistant biofilms, which can substantially impact on patient outcome. In comparison to Candida albicans, C. auris has a lower capacity to form biofilms in in vitro models and a higher capacity when tested on animal skin models. Intraspecies variation is shown to exist, with some clinical isolates having greater biofilm capabilities than others. There is a need for models that closely mimic the real niches where infection occurs on human patients. This protocol describes, in detail, a human skin model to study C. auris biofilm formation using catheterized and non-catheterized skin.

COVID‐19–associated mucormycosis (CAM) has emerged as a challenging complication as the current pandemic has increased the population requiring treatment with corticosteroids. CAM has caused a massive outbreak in India, reported to be causing cases in Iran, Egypt and The Netherlands.

An increasing number of outbreaks due to resistant non-albicans Candida species have been reported worldwide. Between 2014 and 2016, Candida isolates causing invasive candidiasis were recovered in a Mexican hospital. Isolates were identified to species level and antifungal susceptibility was determined. In the time period studied, 74 invasive candidiasis cases were identified, with 38% (28/74) caused by Candida parapsilosis, out of which 54% (15/28) were fluconazole resistant. The ERG11 gene was sequenced for 12 recoverable fluconazole-resistant C. parapsilosis isolates and SNPs identified. The 12 isolates had one common silent point mutation in ERG11 (T591C) and seven isolates had an additional (A395T) mutation, which corresponded to Y132F. Four of the isolates carrying this mutation were closely related within the same cluster by microsatellite typing. This is the first report of an invasive candidiasis outbreak in Mexico due to azole-resistant C. parapsilosis associated with the Y132F substitution.

Malassezia species are a major part of the normal mycobiota and colonize mainly sebum-rich skin regions of the body. This group of fungi cause a variety of infections such as pityriasis versicolor, folliculitis, and fungaemia. In particular, Malassezia sympodialis and its allergens have been associated with non-infective inflammatory diseases such as seborrheic dermatitis and atopic eczema. The aim of this study was to investigate the host response to M. sympodialis on oily skin (supplemented with oleic acid) and non-oily skin using an ex vivo human skin model. Host-pathogen interactions were analyzed by SEM, histology, gene expression, immunoassays and dual species proteomics. The skin response to M. sympodialis was characterized by increased expression of the genes encoding β-defensin 3 and RNase7, and by high levels of S100 proteins in tissue. Supplementation of oleic acid onto skin was associated with direct contact of yeasts with keratinocytes and epidermal damage. In oily conditions, there was increased expression of IL18 but no expression of antimicrobial peptide genes in the skin’s response to M. sympodialis. In supernatants from inoculated skin plus oleic acid, TNFα, IL-6, and IL1-β levels were decreased and IL-18 levels were significantly increased.

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID‐19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS‐CoV‐2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS‐CoV‐2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS‐CoV‐2, with the majority of solutions being effective at high‐working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS‐CoV‐2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.

Background Increasing global incidence of serious fungal infections (SFI) requires increasing access to high quality medical mycology education to improve their identification and decrease associated mortality. Methods An online course was developed by two infectious diseases (ID) specialists. The course aimed to improve diagnosis of SFI and was delivered from April-June 2019. The course consisted of 9 online interactive sessions every week. Sessions were streamed by a hospital in Mexico and local participants gather there every week. Other participants connected independently online. Participants took one exam at the start and another after the course was completed. Feedback was collected during the course. A final evaluation of the impact on diagnosis will be collected in October 2019. Results A total of 137 people registered for the course. Registrants were from four different countries, Mexico (126, 92%), Ecuador (9, 6.6%), Australia (1, 0.7%) and Bolivia (1, 0.7%). Mexican participants connected from 15 of the 32 regions (47%) in Mexico and 45 (33%) attended the course at host hospital. Most participants were physicians (76%), 54 were ID specialists and 19 were ID residents. Sixty participants (60/137, 44%) completed the course, with greater completion by those attending in person (33/45, 73%) compared with online attendance (27/92, 29%). The exams results improved 30% after the course. Clinical urgent calls limited attendance. Conclusions This online course allowed a broad geographical participation. Learning as group lead to better completion rates. Recorded sessions will be available on demand and may allow the completion of the course.

Lower respiratory infections are the most important cause of death due to a transmissible disease. We present a case of severe influenza and coccidioidomycosis lung coinfection in a 65-year-old Mexican migrant. This case highlights the challenges that respiratory viruses impose on the diagnosis of fungal infections and on the multidisciplinary management of these infections. In addition, this case shows how medical complications and superinfections could be potentially prevented if flu vaccination is provided.

Human skin fungal infections (SFIs) affect 25% of the world’s population. Most of these infections are superficial. The main limitation of current animal models of human superficial SFIs is that clinical presentation is different between the different species and animal models do not accurately reflect the human skin environment. An ex vivo human skin model was therefore developed and standardised to accurately model SFIs. In this manuscript, we report our protocol for setting up ex vivo human skin infections and report results from a primary superficial skin infection with Trichophyton rubrum, an anthropophilic fungus. The protocol includes a detailed description of the methodology to prepare the skin explants, establish infection, avoid contamination, and obtain high quality samples for further downstream analyses. Scanning electronic microscopy (SEM), histology and fluorescent microscopy were applied to evaluate skin cell viability and fungal morphology. Furthermore, we describe a broad range of assays, such as RNA extraction and qRT-PCR for human gene expression, and protein extraction from tissue and supernatants for proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS). Non-infected skin was viable after 14 days of incubation, expressed genes and contained proteins associated with proliferative, immune and differentiation functions. The macroscopic damage caused by T. rubrum had a similar appearance to the one expected in clinical settings. Finally, using this model, the host response to T. rubrum infection can be evaluated at different levels.