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Introduction Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. Methods and results In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. Conclusions This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. Significance and impact of the study The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.

Aspergillus fumigatus is a worldwide-distributed saprophytic fungus and the major cause of invasive aspergillosis. This fungus can produce two types of melanin-dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. These pigments are considered important resistance mechanisms to stress, as well as virulence factors. The aim of this review is to present the current knowledge of the genetic basis and metabolic pathways of melanin production, their activation, function, and interaction with the host immune system. The DHN-melanin pathway is encoded in a cluster that includes six genes (abr1, abr2, ayg1, arp1, arp2, and pksP/alb1 genes) whose encoded proteins seem to be the origin of the pigment in endosomes. These vesicles are secreted and the pigment is subsequently located in the wall of the conidium beneath the rodlet layer. Unlike DHN-melanin, pyomelanin does not have its own biosynthetic pathway but is related to the activation of the L-tyrosine/L-phenylalanine degradation pathway that includes a cluster of six genes (hppD, hmgX, hmgA, fahA, maiA, and hmgR). Its production is due to the polymerization of homogentisic acid and is linked to conidial germination. Despite the knowledge gained in recent years, further studies will be necessary to confirm the pathways that produce these pigments and their role in the virulence mechanisms of A. fumigatus.

Infections caused by the opportunistic pathogens Scedosporium/Lomentospora are on the rise. This causes problems in the clinic due to the difficulty in diagnosing and treating them. This review collates information published on immune response against these fungi, since an understanding of the mechanisms involved is of great interest in developing more effective strategies against them. Scedosporium/Lomentospora cell wall components, including peptidorhamnomannans (PRMs), α-glucans and glucosylceramides, are important immune response activators following their recognition by TLR2, TLR4 and Dectin-1 and through receptors that are yet unknown. After recognition, cytokine synthesis and antifungal activity of different phagocytes and epithelial cells is species-specific, highlighting the poor response by microglial cells against L. prolificans. Moreover, a great number of Scedosporium/Lomentospora antigens have been identified, most notably catalase, PRM and Hsp70 for their potential medical applicability. Against host immune response, these fungi contain evasion mechanisms, inducing host non-protective response, masking fungal molecular patterns, destructing host defense proteins and decreasing oxidative killing. In conclusion, although many advances have been made, many aspects remain to be elucidated and more research is necessary to shed light on the immune response to Scedosporium/Lomentospora. This research was funded by the Basque Government, grant number IT1362-19. L.M.-S., M.A., and L.A.-F. were funded by the Basque Government.

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%–100% and 93%–99%, respectively, depending on the cut-off value chosen (four values were proposed A450nm= 0.5837, A450nm= 0.6042, A450nm= 0.6404, and A450nm= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presence.

The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

Background Mucor circinelloides is an opportunistic fungus capable of causing mucormycosis, a highly aggressive infection of quick spreading. Besides, it also has a high mortality rate due to late diagnosis and difficult treatment. Aims In this study we have identified the most immunoreactive proteins of the secretome and the total protein extract of M. circinelloides using sera from immunocompromised infected mice. Methods The proteins of the secretome and the total extract were analyzed by two-dimensional electrophoresis and the most immunoreactive antigens were detected by Western Blot, facing the sera of immunocompromised infected mice to the proteins obtained in both extracts of M. circinelloides. Results Seven antigens were detected in the secretome extract, and two in the total extract, all of them corresponding only to three proteins. The enzyme enolase was detected in both extracts, while triosephosphate isomerase was detected in the secretome, and heat shock protein HSS1 in the total extract. Conclusions In this work the most immunoreactive antigens of the secretome and the total extract of M. circinelloides were identified. The identified proteins are well known fungal antigens and, therefore, these findings can be useful for future research into alternatives for the diagnosis and treatment of mucormycosis.

Candida albicans is a commensal yeast able to cause life threatening invasive infections particularly in immunocompromised patients. Despite the availability of antifungal treatments, mortality rates are still unacceptably high and drug resistance is increasing. We, therefore, generated the Ca37 monoclonal antibody against the C. albicans alcohol dehydrogenase (Adh) 1. Our data showed that Ca37 was able to detect C. albicans cells, and it bound to Adh1 in yeast and Adh2 in hyphae among the cell wall-associated proteins. Moreover, Ca37 was able to inhibit candidal growth following 18h incubation time and reduced the minimal inhibitory concentration of amphotericin B or fluconazole when used in combination with those antifungals. In addition, the antibody prolonged the survival of C. albicans infected-Galleria mellonella larvae, when C. albicans was exposed to antibody prior to inoculating G. mellonella or by direct application as a therapeutic agent on infected larvae. In conclusion, the Ca37 monoclonal antibody proved to be effective against C. albicans, both in vitro and in vivo, and to act together with antifungal drugs, suggesting Adh proteins could be interesting therapeutic targets against this pathogen. Technical and human support provided by the Proteomics Core Facility-SGIker at the UPV/EHU is gratefully acknowledged. We thank the member of the Chartered of Linguists, No 022913 for improving the English in the manuscript. This work was supported by Basque Government (Grant IT1362-19). AA, IB and LMS have received a predoctoral Grant from Basque Government and LAF from UPV/EHU

The high mortality rates of Lomentospora prolificans infections are due, above all, to the tendency of the fungus to infect weakened hosts, late diagnosis and a lack of effective therapeutic treatments. To identify proteins of significance for diagnosis, therapy or prophylaxis, immunoproteomics-based studies are especially important. Consequently, in this study murine disseminated infections were carried out using L. prolificans, Scedosporium aurantiacum, Scedosporium boydii and Aspergillus fumigatus, and their sera used to identify the most immunoreactive proteins of L. prolificans total extract and secreted proteins. The results showed that L. prolificans was the most virulent species and its infections were characterized by a high fungal load in several organs, including the brain. The proteomics study showed a high cross-reactivity between Scedosporium/Lomentospora species, but not with A. fumigatus. Among the antigens identified were, proteasomal ubiquitin receptor, carboxypeptidase, Vps28, HAD-like hydrolase, GH16, cerato-platanin and a protein of unknown function that showed no or low homology with humans. Finally, Hsp70 deserves a special mention as it was the main antigen recognized by Scedosporium/Lomentospora species in both secretome and total extract. In conclusion, this study identifies antigens of L. prolificans that can be considered as potential candidates for use in diagnosis and as therapeutic targets and the production of vaccines. This research was funded by the Basque Government, grant number IT1362-19. I.B., L.M.S. and L.A.F. received a predoctoral fellowship from the Basque Government.

Many studies have analyzed relationships between microorganisms and cancer, demonstrating that microorganisms are able to prevent the onset of cancer and, others to provoke it. Specifically, more and more scientific articles are publishing on microorganisms, linking them to the creation, implementation and dispersion of cancer. In fact, it is estimated that microorganisms cause 17.8% of all cancers. The cancer-causing viral capability is the most studied and, in consequence, many different viral mechanisms that can cause cancer have been described. The International Cancer Re-search Institute has categorized eight viruses for the first time as "carcinogenic to hu-mans", including a human papillomavirus, two herpesvirus and two hepatitis viruses. Regarding bacteria, among cancerous agents, Helicobacter pylori is the most studied in relation to stomach cancer. In addition, many other bacteria, such as Salmonella typhi, Chlamydia pneumoniae and Streptococcus bovis, have been directly related to cancer. Although relatively little research on the effect of fungi on cancer has been investi-gated, some of the toxins produced by these microorganisms have been shown to cause cancer. In addition, some mechanism for the generation and spread of cancer have been described in Candida albicans. Studies to date have shown the influence of microor-ganisms on the development and promotion of cancer. For this reason, to face cancer in the next future, deepen into the relationship between cancer and microorganisms will be essential; Ikerketa askok mikroorganismoen eta minbizien arteko erlazioak aztertu dituzte, eta erakutsi dute mikroorganismo batzuek minbiziaren agerpena saihesten dutela eta beste batzuek, aldiz, minbizia eragin dezaketela. Hain zuzen ere, gero eta artikulu zientifiko gehiago argitaratzen ari dira mikroorganismoak minbiziaren sortzearekin, ezarpenarekin eta sakabanaketarekin erlazionatuz. Izan ere, mikroorganismoek minbizi guztien % 17,8 eragiten dutela estimatu da. Minbizia sortzeko birusen gaitasuna da gehien ikertu dena eta, ondorioz, minbizia sor dezaketen mekanismo desberdin asko deskribatu dira. Minbizia Ikertzeko Nazioarteko Agentziak zortzi birus 1. mailako "gizakiontzat kartzinogeno"-tzat sailkatu ditu; haien artean, giza papiloma birusa, bi herpesbirus eta bi hepatitisaren birus aurkitzen dira. Bakterioei dagokienez, minbizi-eragileen artean, Helicobacter pylori da gehien ikertu dena urdaileko minbiziarekin erlazionatuta. Baina honetaz gain, beste hainbat bakterio, hala nola Salmonella typhi, Chlamydia pneumoniae eta Streptococcus bovis minbiziarekin zuzenki erlazionatu dira. Onddoek daukaten minbiziarekiko erlazioa oso gutxi ikertu den arren, mikroorganismo hauek sortutako toxina batzuek minbizia eragin dezaketela frogatu da. Horrez gain, Candida albicans onddoak minbiziaren sorrera eta hedapena eragin dezakeen hainbat mekanismo deskribatu dira. Orain arte egindako ikerketek mikroorganismoek minbiziaren garapenean eta sustapenean daukaten eragina agerrarazi dute. Hori dela eta, etorkizunean minbiziari aurre egiteko, minbiziaren eta mikroorganismoen arteko erlazioan sakontzea ezinbestekoa da.

The number of fungal isolates resistant to antifungal drugs has increased dramatically over the last few years and has become an important concern for clinicians. Among these isolates, fungi showing multidrug resistance are particularly worrying because of the difficulties associated with their treatment. These factors hamper the successful recovery of patients and drastically raise mortality rates. Antifungal resistance is multifactorial and several mechanisms in different fungi have been described. There is a need to study these mechanisms in depth; however, the study of antifungal drug resistance separately for each individual species makes progress in the field very slow and tedious. The selection of a multiresistant microorganism as a model for understanding resistance mechanisms and extrapolating the results to other species could help in the search for a solution. In this mini-review, we describe the pathobiology of Lomentospora (Scedosporium) prolificans, paying special attention to its intrinsic resistance to all currently available antifungal agents. The characteristics of L. prolificans offer several advantages: the possibility of using a single microorganism for the study of resistance to different drugs, even cases of double and triple resistance; it is biologically safe for society in general as no new genetically-modified strains are needed for the experiments; it is homologous with other fungal species, and there is repetitiveness between different strains. In conclusion, we propose L. prolificans as a candidate for consideration as a fungal model for the study of resistance mechanisms against antifungal agents.